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Sample records for calmodulin peptide 76-121aa

  1. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

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    Cyr Janet L

    2002-11-01

    Full Text Available Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3 in its neck region; we identified a fourth IQ domain (IQ4, located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. Conclusions We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

  2. Clicked bis-PEG-peptide conjugates for studying calmodulin-Kv7.2 channel binding

    OpenAIRE

    Bonache de Marcos, María Ángeles; Alaimo, Alessandro; Malo, Covadonga; Millet, Oscar; Villarroel, Alvaro; González-Muñiz, Rosario

    2014-01-01

    The recombinant Kv7.2 calmodulin (CaM) binding site (Q2AB CaMBD) shows a high tendency to aggregate, thus complicating biochemical and structural studies. To facilitate these studies we have conceived bis-PEG-peptide CaMBD-mimetics linking helices A and B in single, easy to handle molecules. Short PEG chains were selected as spacers between the two peptide molecules, and a Cu(i)-catalyzed cycloaddition (CuAAC) protocol was used to assemble the final bis-PEG-peptide conjugate, by the convenien...

  3. Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies.

    Science.gov (United States)

    Kuczera, Krzysztof; Kursula, Petri

    2012-01-01

    We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the

  4. Photounbinding of calmodulin from a family of CaM binding peptides.

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    Klaus G Neumüller

    Full Text Available BACKGROUND: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. METHODOLOGY/PRINCIPAL FINDINGS: We present studies of the photounbinding of labeled calmodulin (CaM from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. CONCLUSIONS/SIGNIFICANCE: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.

  5. Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System.

    Science.gov (United States)

    Ishida, Hiroaki; Nguyen, Leonard T; Gopal, Ramamourthy; Aizawa, Tomoyasu; Vogel, Hans J

    2016-09-01

    In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells. PMID:27502305

  6. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

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    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  7. Melittin binding causes a large calcium-dependent conformational change in calmodulin.

    OpenAIRE

    Kataoka, M.(LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France); Head, J F; Seaton, B A; Engelman, D M

    1989-01-01

    The interaction between calmodulin and its target protein is a key step in many calcium-regulated cellular functions. Melittin binds tightly to calmodulin in the presence of calcium and is a competitive inhibitor of calmodulin function. Using melittin as a model for the target peptide of calmodulin, we have found a large Ca2+-dependent conformational change of calmodulin in solution induced by peptide binding. Mg2+ does not substitute for Ca2+ in producing the conformation change. Small-angle...

  8. A many-body model to study proteins. II. Incidence of many-body polarization effects on the interaction of the calmodulin protein with four Ca2+ dications and with a target enzyme peptide

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    Cuniasse, Philippe; Masella, Michel

    2003-07-01

    The origin of the interactions occurring in the calmodulin protein interacting with one of its target peptide and counterions, and binding four calcium dications, has been investigated in the gas phase, using the many-body model presented in Paper I [Masella and Cuniasse, J. Chem. Phys. 119, 1866 (2003)] and a classical pairwise force field. As compared to the latter force field, the many-body model is shown to provide a geometrical description of the calmodulin/target peptide structure in better agreement with the x-ray experimental one, and a better description of the Ca2+ binding sites (as compared to "small molecule" structures reported in the Cambridge Structural Database). Regarding the energy, both models provide qualitatively a similar description of the interactions occurring in the calmodulin/target peptide system. However, quantitatively, the pairwise model predicts interaction energies greater by about 25% as compared to the many-body one in the case of calmodulin/Ca2+ interactions. This is due to the inability of pairwise force fields to account for the strong anticooperative effects predicted to occur in [Ca,(carboxylate)n]2-n systems by both the many-body model and quantum computations. Hence, the new many-body model appears to be well suited for describing proteinic systems interacting with cations, both in terms of geometry and energy.

  9. Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.

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    Dan Zhang

    Full Text Available BACKGROUND: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. METHODOLOGY/PRINCIPAL FINDINGS: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR or Catestatin (CAT induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate, a store operated channels (SOCs blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. CONCLUSIONS/SIGNIFICANCE: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated i

  10. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  11. Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure

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    Hayashi Nobuhiro

    2008-02-01

    Full Text Available Abstract Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure

  12. Mutations in calmodulin cause ventricular tachycardia and sudden cardiac death

    DEFF Research Database (Denmark)

    Nyegaard, Mette; Overgaard, Michael Toft; Sondergaard, M.T.;

    2012-01-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause...... a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe...... calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac...

  13. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1994-01-01

    The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability...... insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)...

  14. Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

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    Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

    1996-01-01

    A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

  15. Anti-calmodulins and tricyclic adjuvants in pain therapy block the TRPV1 channel.

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    Zoltán Oláh

    Full Text Available Ca(2+-loaded calmodulin normally inhibits multiple Ca(2+-channels upon dangerous elevation of intracellular Ca(2+ and protects cells from Ca(2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+-uptake via the vanilloid inducible Ca(2+-channel/inflamatory pain receptor 1 (TRPV1, which suggests that calmodulin inhibitors may block pore formation and Ca(2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45Ca(2+-uptake at microM concentrations: calmidazolium (broad range > or = trifluoperazine (narrow range chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially. Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+-uptake in intact TRPV1(+ cells, and suggests an extracellular site of inhibition. TRPV1(+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2

  16. Anti-calmodulins and tricyclic adjuvants in pain therapy block the TRPV1 channel.

    Science.gov (United States)

    Oláh, Zoltán; Jósvay, Katalin; Pecze, László; Letoha, Tamás; Babai, Norbert; Budai, Dénes; Otvös, Ferenc; Szalma, Sándor; Vizler, Csaba

    2007-06-20

    Ca(2+)-loaded calmodulin normally inhibits multiple Ca(2+)-channels upon dangerous elevation of intracellular Ca(2+) and protects cells from Ca(2+)-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+). Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+)-uptake via the vanilloid inducible Ca(2+)-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca(2+) entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45)Ca(2+)-uptake at microM concentrations: calmidazolium (broad range) > or = trifluoperazine (narrow range) chlorpromazine/amitriptyline>fluphenazine>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+) or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+)-uptake in intact TRPV1(+) cells, and suggests an extracellular site of inhibition. TRPV1(+), inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+)-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+)-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+)-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2

  17. Role of Calmodulin in Cell Proliferation

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    Chafouleas, J.

    1983-01-01

    Calmodulin levels were found to increase as cells enter plateau. The data suggest that the cells are exiting the cell cycle late in the G sub 1 phase, or that the calmodulin levels in plateau cells are uncoupled to progression into S phase in plateau cells. Upon release, calmodulin levels rapidly decrease. Following this decrease, there is a increase prior to S phase.

  18. Calmodulin Binding Proteins and Alzheimer's Disease.

    Science.gov (United States)

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  19. Real—time Analysis of the Interaction between Calmodulin and Melittin by SPR Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    WeiGuoLI; XiaoQiangCUI; 等

    2002-01-01

    The dynamic interaction process of calmodulin with an immobilized peptide melittin was investigated in real time by surface plasmon resonance spectroscopy, and dissociation constant of the complex was calculated to be 3.37×10-6 mol/L.

  20. Real-time Analysis of the Interaction between Calmodulin and Melittin by SPR Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The dynamic interaction process of calmodulin with an immobilized peptide melittin was investigated in real time by surface plasmon resonance spectroscopy, and dissociation constant of the complex was calculated to be 3.37′10-6 mol/L.

  1. Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

    International Nuclear Information System (INIS)

    An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. The epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or toponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase

  2. Conformational heterogeneity of the calmodulin binding interface

    Science.gov (United States)

    Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

    2016-04-01

    Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

  3. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Barreda, Elena Gomez de [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Avila, Jesus, E-mail: javila@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); CIBER de Enfermedades Neurodegenerativas, 28031 Madrid (Spain)

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  4. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    SUN; Daye(

    2001-01-01

    [1]Cheng. W. Y., Cyclic 3', 5'-nucleotide phosphodiestrase: demonstration of an activator, Biochm. Biophys. Res. Commun.,1970, 38: 533-538.[2]Boynton, A. L., Whitfield, J. F., MacManus, J. P., Calmodulin stimulates DNA synthesis by rat liver cells, BBRC.1980,95(2): 745-749.[3]Gorbacherskaya, L. V., Borovkova, T. V., Rybin, U. O. et al., Effect of exogenous calmodulin on lymphocyte proliferation in normal subjects, Bull Exp. Med. Biol., 1983, 95: 361-363.[4]Wong, P. Y.-K., Lee, W. H., Chao, PH.-W., The role of calmodulin in prostaglandin metabolism, Ann. NY Acad. Sci.,1980, 356: 179-189.[5]Mac Neil, S., Dawson, R. A., Crocker, G. et al., Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth, J. Invest. Dermatol., 1984, 83: 15-19.[6]Crocker, D. G., Dawson, R. A., Mac Neil, S. et al., An extracellular role for calmodulin-like activity in cell proliferation,Biochem. J., 1988, 253: 877-884.[7]Polito. V. S., Calmodulin and calmodulin inhibitors: effect on pollen germination and tube growth, in Pollen: Biology and Implications for Plant Breeding (eds. Mulvshy, D. L., Ottaviaro, E.), New York: Elsevier, 1983.53-60.[8]Biro, R. L., Sun, D. Y., Roux, S. J.et al., Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution, Plant Physiol., 1984,75: 382-386.[9]Terry, M. E., Bonner, B. A., An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of IAA-induced growth, Plant Physiol., 1980, 66: 321-325.[10]Josefina, H. N., Aldasars, J. J., Rodriguez, D., Localization of calmodulin on embryonic Cice aricium L, in Molecular and Cellular Aspects of Calcium in Plant Development (ed. Trewavas, A. J.), New York, London: Plenum Press, 1985, 313.[11]Dauwalder, M., Roux, S. J., Hardison, L., Distribution of calmodulin in pea seedling: immunocytochemical localization in plumules and root apices, Planta, 1986, 168: 461

  5. Purification, crystallization and preliminary crystallographic studies of a calmodulin-OLFp hybrid molecule

    International Nuclear Information System (INIS)

    The hybrid moelcule of calmodulin and calmodulin-binding domain of olfactory nucleotide-gated ion-channel peptide (CaM-OLFp) was crystallized and preliminary analyzed using X-ray diffaction. A hybrid molecule consisting of calmodulin (CaM) and the CaM-binding domain of olfactory nucleotide-gated ion-channel peptide (CaM-OLFp) was purified and crystallized by the hanging-drop vapour-diffusion method at 298 K. The crystals diffracted to a maximum resolution of 1.85 Å at cryogenic temperature (100 K) using X-rays from a rotating anode (Cu, wavelength 1.54 Å). The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 64.76, b = 36.23, c = 70.96 Å, α = γ = 90, β = 109.4°. Analysis of the packing density shows that the asymmetric unit contains one CaM-OLFp hybrid molecule with a solvent content of 36.42%

  6. Immunoelectron microscopic localization of calmodulin in corn root cells

    Institute of Scientific and Technical Information of China (English)

    LIJIAXU; JIEWENLIU; DAYESUN

    1993-01-01

    Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.

  7. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  8. Neutron and x-ray scattering studies of the interactions between Ca{sup 2+}-binding proteins and their regulatory targets: Comparisons of troponin C and calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J.; Olah, G.A.

    1993-11-01

    The regulatory proteins calmodulin and troponin C share a strikingly unusual overall structure. Their crystal structures show each protein consists of two structurally homologous globular domains connected by an extended, solvent exposed alpha-helix of = 8 turns. Calmodulin regulates a variety of enzymes that show remarkable functional and structural diversity. This diversity extends to the amino acid sequences of the calmodulin-binding domains in the target enzymes. In contrast with calodulin, troponin C appears to have a single very specialized function. It is an integral part of the troponin complex, and Ca{sup 2+} binding to troponin c results in the release of the inhibitory function of troponin I, which eventually leads to actin-binding to myosin and the triggering of muscle contraction. Small-angle scattering has been particularly useful for studying the dumbbell shaped proteins because the technique is very sensitive to changes in the relative dispositions of the two globular domains. Small-angle scattering, using x-rays or neutrons, gives information on the overall shapes of proteins in solution. Small-angle scattering studies of calmodulin and its complexes with calmodulin-binding domains from various target enzymes have played an important role in helping us understand the functional role of its unusual solvent exposed helix. Likewise, small-angle scattering has been used to study troponin C with various peptides, to shed light on the similarities and differences between calmodulin and troponin C.

  9. Calmodulin Binding Proteins and Alzheimer’s Disease

    Science.gov (United States)

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  10. Kv7 channels can function without constitutive calmodulin tethering.

    Directory of Open Access Journals (Sweden)

    Juan Camilo Gómez-Posada

    Full Text Available M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC, a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.

  11. Kv7 Channels Can Function without Constitutive Calmodulin Tethering

    Science.gov (United States)

    Alberdi, Araitz; Alaimo, Alessandro; Etxeberría, Ainhoa; Fernández-Orth, Juncal; Zamalloa, Teresa; Roura-Ferrer, Meritxell; Villace, Patricia; Areso, Pilar; Casis, Oscar; Villarroel, Alvaro

    2011-01-01

    M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function. PMID:21980481

  12. Structural basis for activation of calcineurin by calmodulin

    OpenAIRE

    Rumi-Masante, Julie; Rusinga, Farai I.; Lester, Terrence E.; Dunlap, Tori B.; Williams, Todd D.; Dunker, A. Keith; Weis, David D.; Trevor P Creamer

    2011-01-01

    The highly conserved phosphatase calcineurin plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin. Calmodulin binds to a regulatory domain within calcineurin, causing a conformational change that displaces an autoinhibitory domain from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which calmodulin acti...

  13. Action of pinaverium bromide on calmodulin-regulated functions.

    Science.gov (United States)

    Wuytack, F; De Schutter, G; Casteels, R

    1985-08-01

    Pinaverium bromide at concentrations below 10(-5) M did not inhibit calmodulin-dependent enzymes such as phosphodiesterase and the Ca transport ATPase of the plasma membrane. At higher concentrations the compound interacted with the stimulation of those enzymes by calmodulin and also inhibited the calmodulin-independent activity. A similar inhibitory action was observed for the NaK ATPase. It is concluded that the inhibitory action of pinaverium bromide on smooth muscle concentration at concentrations below 10(-5) M was due to its interaction with the voltage-dependent Ca channels and not to its interference with the calmodulin-dependent activation of the contractile proteins. PMID:2995077

  14. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Science.gov (United States)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  15. The Effect of Calcium on the Binding of Calmodulin to Calcium/Calmodulin Protein Kinase II.

    Science.gov (United States)

    Porta, Angela R.

    2000-01-01

    Introduces a follow-up laboratory experiment demonstrating the formation change when calcium binds to calmodulin. This conformation change allows this complex to bind to a target protein. Presents the necessary information to conduct the experiment and discusses the results. (YDS)

  16. Calmodulin binds to and inhibits the activity of phosphoglycerate kinase.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells. PMID:15363631

  17. Chronic amphetamine treatment increases striatal calmodulin in rats

    International Nuclear Information System (INIS)

    A radioimmunoassay was developed to measure calmodulin in striatum from rats treated with one dose or repeated injections of amphetamine. Chronic, but not acute, amphetamine treatment resulted in a significant increase in total calmodulin levels in striatal homogenates. This effect may be linked to the behavioral sensitization which develops after chronic amphetamine treatments. (Auth.)

  18. Acidic/IQ Motif Regulator of Calmodulin*

    OpenAIRE

    Putkey, John A.; Waxham, M. Neal; Gaertner, Tara R.; Brewer, Kari J.; Goldsmith, Michael; Kubota, Yoshihisa; Kleerekoper, Quinn K.

    2007-01-01

    The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca2+ binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca2+ binding to sites III and IV, and we present a model showing that this could increase Ca2+ binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ ...

  19. Mediation of flowering by a calmodulin-dependent proteinkinase

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A calmodulin-dependent protein kinase (MCK1) appeared important in regulating flowering in tobacco. The expression of modified MCK1 that lacks the C-terminal including calmodulin-binding domain upsets the flowering developmental program, leading to the abortion of flower primordia initiated on the main axis of the plant and, as well, caused the prolongation of the vegetative phase in axillary buds. The abortion process of flowers began first in the developing anthers and subsequently the entire flower senesces. In axillary buds the prolonged vegetative phase was characterized by atypical elongated, narrow, twisted leaves. These results suggested a role for calmodulin-dependent protein kinase homologs in mediating flowering.

  20. Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

    Institute of Scientific and Technical Information of China (English)

    Zi Fang; Zhenguang Yan; Shuo Li; Qin Wang; Weizhong Cao; Guangrui Xu; Xunhao Xiong; Liping Xie; Rongqing Zhang

    2008-01-01

    Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinct-ada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata.

  1. Interaction of smooth muscle relaxant drugs with calmodulin and cyclic nucleotide phosphodiesterase.

    Science.gov (United States)

    Ronca-Testoni, S; Hrelia, S; Hakim, G; Rossi, C A

    1985-01-15

    Some smooth muscle relaxant drugs with an unknown mechanism of action have been tested for their interaction with calmodulin and with calmodulin-induced cyclic nucleotide phosphodiesterase (PDE) activity. The affinity of these drugs for calmodulin does not parallel their inhibitory effect on the calmodulin activation of PDE. The lack of parallelism could be due to a binding of the drugs to different sites on calmodulin; furthermore a binding of papaverine, octylonium bromide and felodipine to PDE molecule might also be considered to explain their inhibitory effect on PDE basal activity. The myolytic effect of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems. PMID:2981701

  2. Acute inhibition of corticosteroidogenesis by inhibitors of calmodulin action.

    Science.gov (United States)

    Carsia, R V; Moyle, W R; Wolff, D J; Malamed, S

    1982-11-01

    To identify the possible role of calmodulin in ACTH function, we tested the ability of chlorpromazine (CP) and other calmodulin antagonists to inhibit steroidogenesis of isolated adrenocortical cells of the rat. CP reversibly inhibited maximal ACTH-induced corticosterone (B) production. The presence of the drug did not alter the ED50 of ACTH stimulation (3.2 X 10(3) pg/ml), suggesting that it inhibited ACTH-induced steroidogenesis in a noncompetitive manner. The CP concentration required for half-maximal inhibition was 8.2 microM, a value close to the dissociation constant of the CP-calmodulin complex (5.3 microM). Concentrations greater than 40 microM resulted in complete inhibition. Similar concentrations of CP inhibited ACTH-induced cAMP accumulation in a dose-dependent manner, indicating an effect of the drug on early events in ACTH action. In addition, CP also apparently acted at a site distal to the point of cAMP formation, as shown by the finding that it inhibited cAMP-induced B production. CP inhibition of ACTH-induced B production was independent of the Ca2+ concentration, suggesting that the drug did not compete with Ca2+ directly. Concentrations of CP greater than 20 microM inhibited protein synthesis as measured by leucine incorporation into cellular proteins. Thus, although the inhibitory effect of high concentrations of CP on steroidogenesis might be explained by an effect on protein synthesis, the inhibition seen at 10 microM appeared to be independent of protein synthesis. Other antagonists of calmodulin action inhibited maximal ACTH-induced B production with the following relative potencies: trifluoperazine greater than CP greater than haloperidol greater than chlordiazepoxide. This order is similar to that reported for inhibition of calmodulin-activated phosphodiesterase and for binding to calmodulin. These findings suggest that calmodulin may modulate the effect of ACTH on steroidogenesis at multiple sites.

  3. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    孙大业; 唐文强; 马力耕

    2001-01-01

    Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca2+ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation,and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore,we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein,phospholipase C, IP3, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.

  4. Calmodulin modulation of ion channels and receptors

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Ion channels and receptors are the structural basis for neural signaling and transmission. Recently, the function of ion channels and receptors has been demonstrated to be modulated by many intracellular and extracellular chemicals and signaling molecules. Increasing evidence indicates that the complexity and plasticity of the function of central nervous system is determined by the modulation of ion channels and receptors. Among various mechanisms, Ca 2+ signaling pathways play important roles in neuronal activity and some pathological changes. Ca 2+ influx through ion channels and receptors can modulate its further influx in a feedback way or modulate other ion channels and receptors. The common feature of the modulation is that Ca 2+ /calmodulin (CaM) is the universal mediator. CaM maintains the coordination among ion channels/receptors and intracellular Ca 2+ homeostasis by feedback modulation of ion channels/receptors activity. This review focuses on the modulating processes of ion channels and receptors mediated by CaM, and further elucidates the mechanisms of Ca 2+ signaling.

  5. Pivoting between calmodulin lobes triggered by calcium in the Kv7.2/calmodulin complex.

    Science.gov (United States)

    Alaimo, Alessandro; Alberdi, Araitz; Gomis-Perez, Carolina; Fernández-Orth, Juncal; Bernardo-Seisdedos, Ganeko; Malo, Covadonga; Millet, Oscar; Areso, Pilar; Villarroel, Alvaro

    2014-01-01

    Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca(2+). First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using (15)N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca(2+) the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca(2+) makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca(2+).

  6. Mediation of flowering by a calmodulin-dependent proteinkinase

    Institute of Scientific and Technical Information of China (English)

    LIANG; Shuping(

    2001-01-01

    [1]Roberts. D. M., Harmon, A. C., Calcium-modulated proteins: Targets of the intracellular signals in higher plants, Ann. Rev.Plant Physiol. Plant Mol. Biol., 1992, 43: 375-414.[2]Sun. D. Y.. Bian, Y. Q., Zhao, B. H. et al., The effects of extracellular calmodulin on cell wall regeneration of protoplasts and cell division, Plant Cell Physiol., 1995, 36: 133-138.[3]Hrabak, E M., Dickmann, L. J., Satterlee, J. S. et al., Characterization of eight new members of the calmodulin-like domain protein kinase gene family from A rabidopsis thaliana, Plant Mol. Biol., 1996, 31:405-412.[4]Huang, J. F., Teyton, L., Harper, J, F., Activation of a Ca2+-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain, Biochemistry, 1996, 35: 13222-13234.[5]Yoo, B. C., Harmon, A. C., Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain, Biochem., 1996, 35: 12029-12037.[6]Saijo, Y., Hata, S., Sheen, J. et al., cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases,Biochem. Biophys. Acta, 1997, 1350: 109-114.[7]Neuhaus. G., Bowler, C., Kern, R. et al., Calcium/calmodulin-dependent and -independent phytochrome signal transduction pathways, Cell, 1993, 73: 937-952.[8]Yang, T., Poovaiah, B. W., Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action, J. Biol. Chem., 2000, 275(5): 3137-3143.[9]Watillon, B., Kettmenn, R., Boxus, P. et al., Calcium/calmodulin-binding serine/threonine protein kinase homologous to mammalian type II calcium/calmodulin-dependent protein kinase is expressed in plant cells, Plant Physiol., 1993, 101:1381-1384.[10]Baum, G., Lev-Yadun, S., Fridmann, Y. et al., Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants, EMBO J, 1996, 15: 2988-2996.[11]Lu, Y. T., Dharmasiri, M. A. N., Harrington

  7. Intracellular levels of calmodulin are increased in transformed cells

    Institute of Scientific and Technical Information of China (English)

    WANG; HONGQINGZHANG; 等

    1992-01-01

    By using Hoechst 33342,rabbit anti calmodulin antibody,FITC-labeled goat anti rabbit IgG and SR101(sulfo rhodamine 101)simultaneously to stain individual normal and transformed cells,the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus,calmodulin and total protein respectively,could be recognized in individualj cells without interference,The phase of the cell cycle was determined by DNA content(Hoechst 33342),We found that in transformed cells(NIH3T3) tsRSV-LA90,cultured at 33℃ and transformed C3H10T1/2 Cells),the ration of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV-LA90 cells,cultured at 39℃ and C3H10T1/2 cells)in every cell cycle phase,This ration increased obviously only from G1 to S phase in either normal or transformed cells.The results showed that calmodulinreally increased during the transformation,and its increase was specific.In the meantime when cells proceeded from G1 to S.the intraceollular calmodulin content also increased specifically.

  8. Bending of the calmodulin central helix : A theoretical study

    NARCIS (Netherlands)

    VanderSpoel, D; DeGroot, BL; Hayward, S; Berendsen, HJC; Vogel, HJ

    1996-01-01

    The crystal structure of calcium-calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is alpha-helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicat

  9. Interaction of a plant pseudo-response regulator with a calmodulin-like protein

    Energy Technology Data Exchange (ETDEWEB)

    Perochon, Alexandre; Dieterle, Stefan; Pouzet, Cecile; Aldon, Didier; Galaud, Jean-Philippe [UMR 5546 CNRS/Universite Toulouse 3, Pole de Biotechnologie vegetale, BP 42617 Auzeville, 31326 Castanet-Tolosan cedex (France); Ranty, Benoit, E-mail: ranty@scsv.ups-tlse.fr [UMR 5546 CNRS/Universite Toulouse 3, Pole de Biotechnologie vegetale, BP 42617 Auzeville, 31326 Castanet-Tolosan cedex (France)

    2010-08-06

    Research highlights: {yields} The pseudo-response regulator PRR2 specifically binds CML9, a calmodulin-like protein {yields} The interaction is confirmed in plant cell nuclei {yields} The interaction requires an intact PRR2 protein. -- Abstract: Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

  10. Involvement of Calmodulin and Calmodulin-like Proteins in Plant Responses to Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    B W Poovaiah

    2015-08-01

    Full Text Available Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM and calmodulin-like proteins (CMLs are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of ROS signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops.

  11. Fluorescence Spectra Studies on the Interaction between Lanthanides and Calmodulin

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    The conformation of Calmodulin(CaM) induced by lanthanides has been examined using fluorescence methods.With the addition of lanthanide (Ln3+), the intrinsic fluorescence intensity of CaM without calcium ions (Apo-CaM) first increases and then decreases.Ln3+ causes the decrease of intrinsic fluorescence intensity of calcium saturated CaM (Ca2+4-CaM) only at high concentrations.At low concentrations, Ln3+ results not only in the enhancement of fluorescence intensity of Apo-CaM, but also in a blue shift of the maximum emission wavelengh of dansyl labeled calmodulin(Apo-D-CaM).The molecular mechanism of the interaction between Ln3+ and CaM has been discussed in the light of the fluorescence spectra.

  12. Calmodulin and calmodulin-binding proteins in cystic fibrosis and normal human fibroblasts

    International Nuclear Information System (INIS)

    The authors have investigated the possibility that a lesion in a calmodulin (CaM)-dependent regulatory mechanism may be involved in cystic fibrosis (CF). The level of CaM, CaM-binding proteins (CaM-BP) and a CaM-dependent phosphatase (CaM-Ptase) have been compared in cultured fibroblasts from CF patients versus age- and sex-matched control subjects. The CaM concentration, measured by radioimmunoassay, ranged from 0.20 to 0.76 μg/mg protein (n=8); there was no significant difference in the average CaM concentration from CF patients vs controls. Using Western blotting techniques with 125I-CaM, they detected at least ten distinct CaM-BPs in fibroblasts with molecular weights ranging from 230K to 37K; the only consistent difference between control and CF cell lines was in a 46.5K CaM-BP, which was depressed in all three CF samples. The 46.5 K CaM-BP was found only in the particulate fraction. A 59K CaM-BP was identified as a CaM-Ptase by its crossreactivity with an antibody against a brain CaM-Ptase. There was no significant difference in CaM-Ptase activity or in the amount of the phosphatase as determined by radioimmunoassay in CF vs. normal samples (n=8). Thus, the level of CaM as well as its various enzymes and proteins do not appear to be altered in CF fibroblasts except for a CaM-BP of 46.5K, the identity of which is currently being investigated

  13. Structures and related properties of helical, disulfide-stabilized peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pagel, M.D. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry]|[Lawrence Berkeley Lab., CA (United States)

    1993-11-01

    The three dimensional structure of several peptides were determined by NMR spectroscopy and distance geometry calculations. Each peptide formed a predictable, rigid structure, consisting of an {alpha}-helix, a {open_quotes}scaffold{close_quotes} region which packed along one face of the helix, and two disulfide bridges which covalently connect the helix and scaffold regions. The peptide Apa-M5 was designed to constrain the M5 peptide from MLCK in a helical geometry using the apamin disulfide scaffold. This scaffold constrains the N- terminal end of the helix with two disulfide bridges and a reverse turn. Like the M5 peptide, Apa-M5 was found to bind calmodulin in a Ca{sup 2+}-dependent 1:1 stoichiometry. However, the dissociation constant of the (Apa-M5)-calmodulin complex, 107 nM, was 100-fold higher than the dissociation constant of the M5-calmodulin complex. This difference was due to a putative steric overlap between the Apa-M5 scaffold and calmodulin. The peptide Apa-Cro was designed to replace the large structural protein matrix of {lambda} Cro with the apamin disulfide scaffold. However, Apa-Cro did not bind the consensus DNA operator half-site of {lambda} Cro, probably due to a steric overlap between the Apa-Cro disulfide framework and the DNA. The amino acid sequence of the scaffold-disulfide bridge arrangement of the peptide Max was derived from the core sequence of scyllatoxin, which contains an {alpha}-helix constrained at the C-terminal end by two disulfide bridges and a two-stranded {beta}sheet scaffold. Max was shown to fold with >84% yield to form a predictable, stable structure that is similar to scyllatoxin. The folding and stability properties of Max make this scaffold and disulfide bridge arrangement an ideal candidate for the development of hybrid sequence peptides. The dynamics of a fraying C-terminal end of the helix of the peptide Apa-AlaN was determined by analysis of {sup 15}N NMR relaxation properties.

  14. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    Science.gov (United States)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  15. Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin.

    Science.gov (United States)

    Lee, Kwangwoon; Alphonse, Sébastien; Piserchio, Andrea; Tavares, Clint D J; Giles, David H; Wellmann, Rebecca M; Dalby, Kevin N; Ghose, Ranajeet

    2016-09-01

    Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells. PMID:27499441

  16. Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

    Institute of Scientific and Technical Information of China (English)

    WANG Mengqiang; MAO Yunxiang; ZHUANG Yunyun; KONG Fanna; SUI Zhenghong

    2009-01-01

    In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensiss,cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exous and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

  17. Cloning and analysis of calmodulin gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

    Science.gov (United States)

    Wang, Mengqiang; Mao, Yunxiang; Zhuang, Yunyun; Kong, Fanna; Sui, Zhenghong

    2009-09-01

    In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

  18. Preparation of Europium Induced Conformation—specific anti—calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    WeiGuoLI; ChaoQI; 等

    2002-01-01

    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  19. Preparation of Europium Induced Conformation-specific anti-calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  20. Nitric Oxide Synthases Reveal a Role for Calmodulin in Controlling Electron Transfer

    Science.gov (United States)

    Abu-Soud, Husam M.; Stuehr, Dennis J.

    1993-11-01

    Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O_2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.

  1. Calmodulin transduces Ca2+ oscillations into differential regulation of its target proteins.

    Science.gov (United States)

    Slavov, Nikolai; Carey, Jannette; Linse, Sara

    2013-04-17

    Diverse physiological processes are regulated differentially by Ca(2+) oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca(2+) binding affinities, and target exchange rates that depend on both target identity and Ca(2+) occupancy. The binding dynamics among Ca(2+), Mg(2+), calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca(2+)-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca(2+) transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin.

  2. Facilitation of plateau potentials in turtle motoneurones by a pathway dependent on calcium and calmodulin

    DEFF Research Database (Denmark)

    Perrier, J F; Mejia-Gervacio, S; Hounsgaard, J

    2000-01-01

    1. The involvement of intracellular calcium and calmodulin in the modulation of plateau potentials in motoneurones was investigated using intracellular recordings from a spinal cord slice preparation. 2. Chelation of intracellular calcium with BAPTA-AM or inactivation of calmodulin with W-7 or tr...

  3. Characterization of a calmodulin binding protein kinase from Arabidopsis thalian

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A full-length calmodulin binding protein kinase cDNA, AtCBK1, from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE. Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests that AtCBK1 encodes a CaM-binding serine/threonine protein kinase.

  4. Isolation and characterization of a novel calmodulin-binding protein from potato

    Science.gov (United States)

    Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

    2002-01-01

    Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

  5. Small-angle x-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly. alpha. -helical conformation

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Mikio; Engelman, D.M. (Yale Univ., New Haven, CT (USA)); Head, J.F. (Boston Univ., MA (USA)); Persechini, A.; Kretsinger, R.H. (Univ. of Virginia, Charlottesville (USA))

    1991-02-05

    Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca{sup 2+}, the observed radii of gyration are 22.4 {angstrom} for wild-type bacterially expressed calmodulin, 19.5 {angstrom} for Des2 calmodulin, and 20.3 {angstrom} for Des4 calmodulin. A reduction in the radius of gyration by 1-2 {angstrom} on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows that the structural properties of the deletion mutants are well described by modifications of an {alpha} helix in the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.

  6. Small-angle x-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly α-helical conformation

    International Nuclear Information System (INIS)

    Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 angstrom for wild-type bacterially expressed calmodulin, 19.5 angstrom for Des2 calmodulin, and 20.3 angstrom for Des4 calmodulin. A reduction in the radius of gyration by 1-2 angstrom on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows that the structural properties of the deletion mutants are well described by modifications of an α helix in the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide

  7. Tracking and localization of calmodulin in live cells.

    Science.gov (United States)

    Johnson, Carey K; Harms, Gregory S

    2016-08-01

    The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from 10μm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells. PMID:27113857

  8. Insulin phosphorylates calmodulin in preparations of solubilized rat hepatocyte insulin receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sacks, D.B.; McDonald, J.M.

    1987-05-01

    It has previously been shown that insulin stimulates the phosphorylation of calmodulin in adipocyte insulin receptor preparations. Here they demonstrate that insulin also stimulates the phosphorylation of calmodulin in wheat germ lectin-enriched insulin receptor preparations obtained from rat hepatocytes. Standard phosphorylation assays were performed at 30C in the presence of 50mM Tris-HCl (pH 7.5), 0.1% (v/v) Triton X-100, 1mM EGTA, 50 M (el-TSP)ATP, 5mM MgCl2, 0.25 M polylysine, 1.2 M calmodulin and various CaS and insulin concentrations. The phosphorylation of calmodulin was determined by SDS-PAGE and autoradiography. Phosphorylation of calmodulin had an absolute requirement for insulin receptors, insulin and certain basic proteins. Phosphorylation was maximal above 13 nM insulin and at submicromolar CaS concentrations, whereas supramicromolar CaS concentrations were inhibitory. As was observed in the adipocyte insulin receptor system, calmodulin phosphorylation was dependent upon the presence of co-factors, such as polylysine, histone H/sub f/2b and protamine sulfate. The role played by these co-factors has not yet been established. These data suggest that both CaS and calmodulin participate in post receptor insulin events in hepatocytes.

  9. Heparin blocks /sup 125/I-calmodulin internalization by isolated rat renal brush border membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meezan, E.; Elgavish, A.; Roden, L.; Wallace, R.W.

    1986-03-05

    /sup 125/I-Calmodulin is internalized by isolated rat renal brush border membrane vesicles (BBV) in a time, temperature and calcium dependent manner. Internalization of /sup 125/I-calmodulin into the osmotically sensitive space of BBV was distinguished from binding of the ligand to the outer BBV surface by examining the interaction of ligand and BBV at different medium osmolarities (300-1100 mosm), uptake was inversely proportional to medium osmolarity. Internalized /sup 125/I-calmodulin was intact and Western blots of solubilized BBV with /sup 125/I-calmodulin demonstrated the presence of several calmodulin-binding proteins of 143, 118, 50, 47.5, 46.5 and 35 kilodaltons which could represent potential intravesicular binding sites for the ligand. Heparin and the related glycosaminoglycan heparin sulfate both showed a dose-dependent inhibition (0.5-50 ..mu..g/ml) of /sup 125/I-calmodulin uptake by BBV, but other sulfated and nonsulfated glycosaminoglycans including chondroitin sulfates, keratan sulfate and hyaluronic acid showed little or no inhibitory effect. Desulfation of heparin virtually abolished the inhibition of uptake while depolymerization reduced it. Heparin did not block the binding of /sup 125/I-calmodulin to BBV proteins as assessed by Western blotting technique suggesting its effect was on internalization of the ligand rather than on its association with internal membrane proteins.

  10. Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

    Science.gov (United States)

    Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

    2000-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

  11. Identification of the Calmodulin-Binding Domains of Fas Death Receptor.

    Directory of Open Access Journals (Sweden)

    Bliss J Chang

    Full Text Available The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD and that of the Fas-associated protein (FADD interact to form the core of the death-inducing signaling complex (DISC, a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD. However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR, biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1 and 251-288 (Fas-Pep2 constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling

  12. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

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    Rodriguez-del Valle Nuri

    2007-11-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

  13. Calmodulin affects sensitization of Drosophila melanogaster odorant receptors

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    Latha eMukunda

    2016-02-01

    Full Text Available Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs, occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX and a highly conserved co-receptor protein (Orco. The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs expressing Or22a inside the fly’s antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native

  14. Cooperative phenomena in binding and activation of Bordetella pertussis adenylate cyclase by calmodulin.

    Science.gov (United States)

    Bouhss, A; Krin, E; Munier, H; Gilles, A M; Danchin, A; Glaser, P; Bârzu, O

    1993-01-25

    The catalytic domain of Bordetella pertussis adenylate cyclase located within the first 400 amino acids of the protein can be cleaved by trypsin in two subdomains (T25 and T18) corresponding to ATP-(T25) and calmodulin (CaM)-(T18) binding sites. Reassociation of subdomains by CaM is a cooperative process, which is a unique case among CaM-activated enzymes. To understand better the molecular basis of this phenomenon, we used several approaches such as partial deletions of the adenylate cyclase gene, isolation of peptides of various size, and site-directed mutagenesis experiments. We found that a stretch of 72 amino acid residues overlapping the carboxyl terminus of T25 and the amino terminus of T18 accounts for 90% of the binding energy of adenylate cyclase-CaM complex. The hydrophobic "side" of the helical region situated around Trp242 plays a major role in the interaction of adenylate cyclase with CaM, whereas basic residues that alternate with acidic residues in bacterial enzyme play a much less important role. The amino-terminal half of the catalytic domain of adenylate cyclase contributes only 10% to the binding energy of CaM, whereas the last 130 amino acid residues are not at all involved in binding. However, these segments of adenylate cyclase might affect protein/protein interaction and catalysis by propagating conformational changes to the CaM-binding sequence which is located in the middle of the catalytic domain of bacterial enzyme. PMID:8420945

  15. Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes

    DEFF Research Database (Denmark)

    Ye, Q; Wei, Y; Fischer, R;

    1997-01-01

    Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for protein...... the most pronounced alterations. In contrast, CaM protein levels were increased in all these cell lines as determined by a radioimmunoassay. These results suggest that oncogenic up-regulation of CaM synthesis takes place posttranscriptionally. Several CaM binding proteins were found at different...... concentrations in the studied cell lines depending on the oncogenes used for transformation. However, CaM overexpression does not seem to affect the overall levels of CaM binding proteins....

  16. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  17. AKAP150-mediated TRPV1 sensitization is disrupted by calcium/calmodulin

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    Shapiro Mark S

    2011-05-01

    Full Text Available Abstract Background The transient receptor potential vanilloid type1 (TRPV1 is expressed in nociceptive sensory neurons and is sensitive to phosphorylation. A-Kinase Anchoring Protein 79/150 (AKAP150 mediates phosphorylation of TRPV1 by Protein Kinases A and C, modulating channel activity. However, few studies have focused on the regulatory mechanisms that control AKAP150 association with TRPV1. In the present study, we identify a role for calcium/calmodulin in controlling AKAP150 association with, and sensitization of, TRPV1. Results In trigeminal neurons, intracellular accumulation of calcium reduced AKAP150 association with TRPV1 in a manner sensitive to calmodulin antagonism. This was also observed in transfected Chinese hamster ovary (CHO cells, providing a model for conducting molecular analysis of the association. In CHO cells, the deletion of the C-terminal calmodulin-binding site of TRPV1 resulted in greater association with AKAP150, and increased channel activity. Furthermore, the co-expression of wild-type calmodulin in CHOs significantly reduced TRPV1 association with AKAP150, as evidenced by total internal reflective fluorescence-fluorescence resonance energy transfer (TIRF-FRET analysis and electrophysiology. Finally, dominant-negative calmodulin co-expression increased TRPV1 association with AKAP150 and increased basal and PKA-sensitized channel activity. Conclusions the results from these studies indicate that calcium/calmodulin interferes with the association of AKAP150 with TRPV1, potentially extending resensitization of the channel.

  18. Calmodulin Gene Expression in Response to Mechanical Wounding and Botrytis cinerea Infection in Tomato Fruit

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    Hui Peng

    2014-08-01

    Full Text Available Calmodulin, a ubiquitous calcium sensor, plays an important role in decoding stress-triggered intracellular calcium changes and regulates the functions of numerous target proteins involved in various plant physiological responses. To determine the functions of calmodulin in fleshy fruit, expression studies were performed on a family of six calmodulin genes (SlCaMs in mature-green stage tomato fruit in response to mechanical injury and Botrytis cinerea infection. Both wounding and pathogen inoculation triggered expression of all those genes, with SlCaM2 being the most responsive one to both treatments. Furthermore, all calmodulin genes were upregulated by salicylic acid and methyl jasmonate, two signaling molecules involved in plant immunity. In addition to SlCaM2, SlCaM1 was highly responsive to salicylic acid and methyl jasmonate. However, SlCaM2 exhibited a more rapid and stronger response than SlCaM1. Overexpression of SlCaM2 in tomato fruit enhanced resistance to Botrytis-induced decay, whereas reducing its expression resulted in increased lesion development. These results indicate that calmodulin is a positive regulator of plant defense in fruit by activating defense pathways including salicylate- and jasmonate-signaling pathways, and SlCaM2 is the major calmodulin gene responsible for this event.

  19. The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

    OpenAIRE

    Huang, Hao; Ishida, Hiroaki; Vogel, Hans J.

    2010-01-01

    Soybean calmodulin isoform 4 (sCaM4) is a plant calcium-binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo-) form of sCaM4 possesses a half unfolded structure, with the N-terminal domain unfolded and the C-terminal domain folded. This result was unexpected as the apo-forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high...

  20. Is buffer a good proxy for a crowded cell-like environment? A comparative NMR study of calmodulin side-chain dynamics in buffer and E. coli lysate.

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    Michael P Latham

    Full Text Available Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded "cell-like" environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca(2+-bound and Ca(2+, peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed "test-tube" studies, experiments performed under conditions that are "cell-like" are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

  1. Characterization and functional analysis of the calmodulin-binding domain of Rac1 GTPase.

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    Bing Xu

    Full Text Available Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151-164 in Rac1 is essential for calmodulin binding. Within the 151-164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A, activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.

  2. Impact of methionine oxidation on calmodulin structural dynamics

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin [Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455 (United States); Moen, Rebecca J. [Chemistry and Geology Department, Minnesota State University, Mankato, MN 56001 (United States); Olenek, Michael J. [Biology Department, University of Wisconsin, La Crosse, WI 54601 (United States); Klein, Jennifer C., E-mail: jklein@uwlax.edu [Biology Department, University of Wisconsin, La Crosse, WI 54601 (United States); Thomas, David D., E-mail: ddt@umn.edu [Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455 (United States)

    2015-01-09

    Highlights: • We measured the distance distribution between two spin labels on calmodulin by DEER. • Two structural states, open and closed, were resolved at both low and high Ca. • Ca shifted the equilibrium toward the open state by a factor of 13. • Methionine oxidation, simulated by glutamine substitution, decreased the Ca effect. • These results have important implications for aging in muscle and other tissues. - Abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron–electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4 nm (closed) and another at ∼6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each

  3. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    Science.gov (United States)

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  4. Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response calmodulin-binding transcription factor AtSR1/CAMTA3

    Science.gov (United States)

    Calcium/calmodulin (Ca2+/CaM) has long been considered a crucial component in wound signaling pathway. However, no functional significance of Ca2+/CaM-binding proteins has been identified in plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca2+/CaM-bindi...

  5. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    O' Day, Danton H., E-mail: danton.oday@utoronto.ca [Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M5S 3G5 (Canada); Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario, Canada L5L 1C6 (Canada); Huber, Robert J. [Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M5S 3G5 (Canada); Suarez, Andres [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario, Canada L5L 1C6 (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Extracellular calmodulin is present throughout growth and development in Dictyostelium. Black-Right-Pointing-Pointer Extracellular calmodulin localizes within the ECM during development. Black-Right-Pointing-Pointer Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. Black-Right-Pointing-Pointer Extracellular calmodulin exists in eukaryotic microbes. Black-Right-Pointing-Pointer Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca{sup 2+}/CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  6. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  7. Calmodulin kinase II inhibition protects against structural heart disease.

    Science.gov (United States)

    Zhang, Rong; Khoo, Michelle S C; Wu, Yuejin; Yang, Yingbo; Grueter, Chad E; Ni, Gemin; Price, Edward E; Thiel, William; Guatimosim, Silvia; Song, Long-Sheng; Madu, Ernest C; Shah, Anisha N; Vishnivetskaya, Tatiana A; Atkinson, James B; Gurevich, Vsevolod V; Salama, Guy; Lederer, W J; Colbran, Roger J; Anderson, Mark E

    2005-04-01

    Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

  8. Structural basis for activation of calcineurin by calmodulin.

    Science.gov (United States)

    Rumi-Masante, Julie; Rusinga, Farai I; Lester, Terrence E; Dunlap, Tori B; Williams, Todd D; Dunker, A Keith; Weis, David D; Creamer, Trevor P

    2012-01-13

    The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein. PMID:22100452

  9. Structure and expression of the chicken calmodulin I gene

    DEFF Research Database (Denmark)

    Ye, Q; Berchtold, M W

    1997-01-01

    The chicken calmodulin I (CaMI) gene has been isolated and characterized on the level of cDNA and genomic DNA. The deduced amino acid (aa) sequence is identical to the one of chicken CaMII which consists of 148 aa. The CaMI gene contains six exons. Its intron/exon organization is identical...... to that of the chicken CaMII and the CaMI and CaMIII genes of rat and human. Expression of the CaMI gene was detected in all chicken tissues examined, although at varying levels. The gene is transcribed into four mRNAs of 0.8, 1.4, 1.7 and 4.4 kb as determined by Northern blot analysis. Our results demonstrate...... that the "multigene-one-protein" principle of CaM synthesis is not only applicable to mammals whose CaM is encoded by three different genes, but also to chickens....

  10. Calmodulin stimulation of calcium transport in carrot microsomal vesicles

    International Nuclear Information System (INIS)

    ATP-dependent 45Ca2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca2+. These results are consistent with the ER being an important site of intracellular Ca2+ regulation

  11. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  12. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  13. Human platelet calmodulin-binding proteins: identification and Ca/sup 2 +/-dependent proteolysis upon platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1987-05-19

    Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and /sup 125/I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound /sup 125/I-calmodulin in a Ca/sup 2 +/-dependent manner; the binding was blocked by ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca/sup 2 +/ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca/sup 2 +/-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca/sup 2 +/ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca/sup 2 +/. The data indicate that limited proteolysis of Ca/sup 2 +//calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca/sup 2 +/ associated with platelet aggregation.

  14. Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607.

    Science.gov (United States)

    Sharma, S; Giri, S; Khuller, G K

    1998-06-01

    A soluble Ca2+/calmodulin dependent protein kinase has been partially purified (approximately 400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC50 of 1.5 and 0.25 microm respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca2+/calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism. PMID:9655195

  15. Effects of calmodulin and calmodulin inhibitors on Ca uptake by sarcoplasmic reticulum of saponin skinned caudal artery

    International Nuclear Information System (INIS)

    Calmodulin (CaM) stimulates plasma membrane transport in many cell types, however, its role in Ca regulation by the sarcoplasmic reticulum (SR) in smooth muscle has not been established. 45Ca uptake was studied in saponin skinned strips of rat caudal artery as a function of CaM and the CaM inhibitors, W-7, calmidazolium (CaMZ), and trifluoperazine (TFP). Although caudal artery strips lose approximately 30% of total tissue CaM during skinning, 0.3 - 2 μM CaM did not increase 45Ca uptake over a wide range of free Ca concentrations (10-8 - 10-6M). Neither W-7 nor CaMZ at concentration of 10-4 - 2 x 10-4M inhibited the MgATP-dependent Ca uptake. Ca uptake was not affected by 50 μM TFP but a significant inhibition was produced by 500 μM. Studies of the effects of TFP on 45Ca efflux indicated that TFP concentrations which inhibited Ca uptake also significantly increased the rate of Ca release. The results suggest that total Ca uptake in caudal artery depends mainly upon MgATP and is not modulated by exogenous CaM or affected by these CaM inhibitors. They cannot preclude that CaM may affect initial velocities or that the CaM inhibitors failed to reach active sites

  16. Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase in free and calmodulin-bound forms

    International Nuclear Information System (INIS)

    A rabbit lung Ca2+-stimulated cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography in the presence of EGTA on DEAE-cellulose and G-200 Sephadex columns still responded to Ca2+ and contained calmodulin (CaM) suggesting that the enzyme exists as a stable CaM-PDE complex. A similar enzyme was demonstrated to exist in bovine lung extract. A monoclonal antibody Cl previously shown to react with the 60 kDa subunit of bovine brain PDE isozymes cross-reacted with the lung enzyme. Purification of the lung enzyme by Cl antibody immunoaffinity chromatography rendered the enzyme dependent of exogenously added CaM for Ca2+ stimulation. The enzyme was further purified by CaM affinity chromatography to near homogeneity. The purified enzyme could be reconstituted into PDE-CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. When reconstitution was carried out in the presence of 45Ca2+, followed by isolation of the protein complex, no 45Ca2+ was found to be associated with the complex. CaM antagonists: trifluoroperazine, compound 48/80 and calcineurin at concentrations abolishing CaM-stimulation of bovine brain PDE had little effect on the bovine lung PDE-CaM complex

  17. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Science.gov (United States)

    Li, Xing-Xia; Yu, Wen-Chao; Cai, Zhong-Qiang; He, Cheng; Wei, Na

    2016-01-01

    The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization. PMID:27703977

  18. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Directory of Open Access Journals (Sweden)

    Xing-Xia Li

    2016-01-01

    Full Text Available The shell of the pearl oyster (Pinctada fucata mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

  19. Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts.

    Science.gov (United States)

    Martin, Tamara P; Lawan, Ahmed; Robinson, Emma; Grieve, David J; Plevin, Robin; Paul, Andrew; Currie, Susan

    2014-02-01

    Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts. PMID:23881186

  20. Calmodulin interacts with PAC1 and VPAC2 receptors and regulates PACAP-induced FOS expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Falktoft, B.; Georg, B.; Fahrenkrug, J.

    2009-01-01

    is a well-known marker of neuronal activation, so we used a human neuroblastoma cell line NB-1 to explore the role of calmodulin in PACAP-induced FOS gene expression. We observed both short-term and prolonged altered PACAP-mediated activation of the FOS gene in the presence of the calmodulin-antagonist W-7...

  1. Structural characterization of the interaction of human lactoferrin with calmodulin.

    Directory of Open Access Journals (Sweden)

    Jessica L Gifford

    Full Text Available Lactoferrin (Lf is an 80 kDa, iron (Fe(3+-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf's interaction with calmodulin (CaM, a ubiquitous, 17 kDa regulatory calcium (Ca(2+-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 kDa, TROSY-based NMR techniques were employed to structurally characterize Ca(2+-CaM when bound to intact apo-Lf. Both CaM's backbone amides and the ε-methyl group of key methionine residues were used as probes in chemical shift perturbation and cross-saturation experiments to define the binding interface of apo-Lf on Ca(2+-CaM. Unlike the collapsed conformation through which Ca(2+-CaM binds the CaM-binding domains of its classical targets, Ca(2+-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca(2+-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM's primary binding domain. Secondary interfaces play a key role in the target's response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes.

  2. Matricellular signal transduction involving calmodulin in the social amoebozoan dictyostelium.

    Science.gov (United States)

    O'Day, Danton H; Huber, Robert J

    2013-01-01

    The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa) releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes. PMID:24705101

  3. Matricellular Signal Transduction Involving Calmodulin in the Social Amoebozoan Dictyostelium

    Directory of Open Access Journals (Sweden)

    Danton H. O'Day

    2013-02-01

    Full Text Available The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL repeat-containing, calmodulin (CaM-binding protein (CaMBP CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes.

  4. Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

    International Nuclear Information System (INIS)

    The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca2+/calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis

  5. Orai1 pore residues control CRAC channel inactivation independently of calmodulin.

    Science.gov (United States)

    Mullins, Franklin M; Yen, Michelle; Lewis, Richard S

    2016-02-01

    Ca(2+) entry through CRAC channels causes fast Ca(2+)-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca(2+)-CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl(-) modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation. PMID:26809793

  6. Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Siems, W. F.; Jones, J. P.; Poovaiah, B. W.

    2001-01-01

    The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

  7. Ca2+ binding sites in calmodulin and troponin C alter interhelical angle movements.

    Science.gov (United States)

    Goto, Kunihiko; Toyama, Akira; Takeuchi, Hideo; Takayama, Kazuyoshi; Saito, Tsutomu; Iwamoto, Masatoshi; Yeh, Jay Z; Narahashi, Toshio

    2004-03-12

    Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism. PMID:15013750

  8. Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.

    Directory of Open Access Journals (Sweden)

    Silviya R Stateva

    Full Text Available Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7 inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR, in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

  9. Chromogranin A-derived peptides are involved in innate immunity.

    Science.gov (United States)

    Aslam, R; Atindehou, M; Lavaux, T; Haïkel, Y; Schneider, F; Metz-Boutigue, M-H

    2012-01-01

    New endogenous antimicrobial peptides (AMPs) derived from chromogranin A (CgA) are secreted by nervous, endocrine and immune cells during stress. They display antimicrobial activities by lytic effects at micromolar range using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. These AMPs can also penetrate quickly into neutrophils (without lytic effects), where, similarly to "cell penetrating peptides", they interact with cytoplasmic calmodulin, and induce calcium influx via Store Operated Channels therefore triggering neutrophils activation. Staphylococcus aureus and Salmonella enteritis are bacteria responsible for severe infections. We investigated here the effects of S. aureus and S. enteritis bacterial proteases on CgA-derived peptides and evaluated their antimicrobial activities. We showed that the Glu-C protease produced by S. aureus V8 induces the loss of the AMPs antibacterial activities and produces new antifungal peptides. In addition, four antimicrobial CGA-derived peptides (chromofungin, procatestatin, human/bovine catestatin) are degraded when treated with bacterial supernatants from S. aureus and S. enteritis, whereas, cateslytin, the short active form of catestatin, resists to this degradation. Finally, we demonstrate that several antimicrobial CgA-derived peptides are able to act synergistically with antibiotics against bacteria and fungi indicating their roles in innate defense.

  10. Effect of calmodulin antagonists on contraction and45Ca movements in rat aorta

    NARCIS (Netherlands)

    Wermelskirchen, D.; Koch, P.; Wilhelm, D.; Nebel, U.; Leidig, A.; Wilffert, B.; Peters, Thies

    1989-01-01

    To study the selectivity of calmodulin antagonists it was assumed that they should inhibit noradrenaline (NA)- and K+-induced contractions similarly without an accompanying inhibition of45Ca uptake. Therefore, in isolated rat aorta the effects of W-7, calmidazolium and trifluoperazine on contraction

  11. Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

    DEFF Research Database (Denmark)

    Li, Hongbing; Panina, Svetlana; Kaur, Amandeep;

    2012-01-01

    Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca2+/CaM complexes, which interact with and activate target proteins...

  12. Structural analysis of calmodulin binding to ion channels demonstrates the role of its plasticity in regulation.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Waterbeemd, M. van de; Bokhovchuk, F.M.; Bate, N.; Bindels, R.J.M.; Hoenderop, J.G.J.; Vuister, G.W.

    2013-01-01

    The Ca2+-binding protein calmodulin (CaM) is a well-known regulator of ion-channel activity. Consequently, the Protein Data Bank contains many structures of CaM in complex with different fragments of ion channels that together display a variety of binding modes. In addition to the canonical interact

  13. Ca2+-calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Kiens, Bente; Richter, Erik

    2006-01-01

    Ca2+ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed...

  14. Role of calmodulin (δ-subunit) in activation of phosphorylase kinase from rabbit skeletal muscles

    International Nuclear Information System (INIS)

    The structure of the inactivated and activated forms of phospholyase kinase was compared. The enzyme was activated by incubation in an alkaline medium (pH 8.5), phosphorylation of the catalytic subunit of cAMP-dependent protein kinase, and limited proteolysis. Hydrophobic chromatography on phenyl-Sepharose and electrophoresis in a polyacrylamide gel density gradient were employed for a comparison of these forms of the enzyme. Activation of the enzyme was accompanied by the separation of a low-molecular-weight component (M/sub r/ about 17,000). The low-molecular-weight protein was obtained in a homogeneous state by chromatography on phenyl-Sepharose. It was established that its properties are similar to those of calmodulin. The presence of calmodulin in preparations of phosphorylase kinase was judged by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in much the same way as bovine brain calmodulin. The results obtained suggest that the δ-subunit is a protein inhibitor of the enzyme

  15. ACQUISITION AND LOSS OF NEURONAL CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE DURING NEURONAL DIFFERENTIATION

    Science.gov (United States)

    Neurons display characteristic schedules by which they acquire and lose the neuron-specific Ca2+/calmodulin-dependent protein Kinase-Gr (CaM Kinase-Gr) during differentiation. uch schedules are exemplified by patterns of expression of this kinase in the developing cerebellum and ...

  16. Control of Ca2+ Influx and Calmodulin Activation by SK-Channels in Dendritic Spines.

    Directory of Open Access Journals (Sweden)

    Thom Griffith

    2016-05-01

    Full Text Available The key trigger for Hebbian synaptic plasticity is influx of Ca2+ into postsynaptic dendritic spines. The magnitude of [Ca2+] increase caused by NMDA-receptor (NMDAR and voltage-gated Ca2+ -channel (VGCC activation is thought to determine both the amplitude and direction of synaptic plasticity by differential activation of Ca2+ -sensitive enzymes such as calmodulin. Ca2+ influx is negatively regulated by Ca2+ -activated K+ channels (SK-channels which are in turn inhibited by neuromodulators such as acetylcholine. However, the precise mechanisms by which SK-channels control the induction of synaptic plasticity remain unclear. Using a 3-dimensional model of Ca2+ and calmodulin dynamics within an idealised, but biophysically-plausible, dendritic spine, we show that SK-channels regulate calmodulin activation specifically during neuron-firing patterns associated with induction of spike timing-dependent plasticity. SK-channel activation and the subsequent reduction in Ca2+ influx through NMDARs and L-type VGCCs results in an order of magnitude decrease in calmodulin (CaM activation, providing a mechanism for the effective gating of synaptic plasticity induction. This provides a common mechanism for the regulation of synaptic plasticity by neuromodulators.

  17. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Bokhovchuk, F.M.; Vuister, G.W.

    2012-01-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-t

  18. A dynamic model of interactions of Ca2+, calmodulin, and catalytic subunits of Ca2+/calmodulin-dependent protein kinase II.

    Directory of Open Access Journals (Sweden)

    Shirley Pepke

    2010-02-01

    Full Text Available During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII, are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic

  19. Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2002-05-31

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. PMID:11919178

  20. Modulation of chloroplast movement in the green alga Mougeotia by the Ca2+ ionophore A23187 and by calmodulin antagonists.

    OpenAIRE

    Serlin, B S; Roux, S J

    1984-01-01

    The Ca2+ ionophore A23187 can induce chloroplast rotation within a single nonirradiated Mougeotia cell. The induced turning was dependent on the position of ionophore application and Ca2+ in the external medium. The role of calmodulin in mediating light-induced chloroplast rotation in the alga Mougeotia was investigated by using the paired calmodulin-antagonist drugs W5-W7 and W12-W13. In each pair, the antagonist with the greater affinity for calmodulin had the greater inhibitor effect on...

  1. Antimicrobial peptides.

    Science.gov (United States)

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  2. Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Ali Adem Bahar

    2013-11-01

    Full Text Available The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs, a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics.

  3. Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

    Science.gov (United States)

    Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

    1996-01-01

    A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

  4. Purification and characterization of a Ca2+ -dependent/calmodulin-stimulated protein kinase from moss chloronema cells

    Indian Academy of Sciences (India)

    Jacinta S D’souza; Man Mohan Johri

    2003-03-01

    We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the moss Funaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 M free Ca2+, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Under in vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.

  5. Biosensor-Based Approach Identifies Four Distinct Calmodulin-Binding Domains in the G Protein-Coupled Estrogen Receptor 1

    OpenAIRE

    Tran, Quang-Kim; VerMeer, Mark

    2014-01-01

    The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific liga...

  6. Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    Directory of Open Access Journals (Sweden)

    Sixt SU

    2010-10-01

    Full Text Available Abstract The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved protein 1. Ubiquitylated proteins were degraded by the 26S protea-some in an ATP-depended manner. The degradation of ubiquitylated proteins were controlled by isopeptidase cleavage. A well characterised system of ubiquitylation and deubiquitylation is the calmodulin system in vitro 2. Detection of ubiquityl-calmodulin conjugtates in vivo have not been shown so far. In this article we discuss the detection of ubiquitin calmodulin conjugates in vivo by incubation with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues. Proteins with a molecular weight of ubiquityl-calmodulin conjugates could be detected in all organs tested. Incubation with ubiquitylprotein-isopeptidase showed clearly a decrease of ubiquitin calmodulin conjugates in vivo with an origination of unbounded ubiquitin. These results suggest that only few ubiquitin calmodulin conjugates exist in rabbit tissues.

  7. Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: More evidence for oxidative stress in vitiligo

    International Nuclear Information System (INIS)

    Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10-3 M H2O2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10-3M H2O2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H2O2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H2O2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo

  8. Calmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis.

    Science.gov (United States)

    O'Day, Danton H; Myre, Michael A

    2004-08-01

    The calcium hypothesis of Alzheimer's disease (AD) invokes the disruption of calcium signaling as the underlying cause of neuronal dysfunction and ultimately apoptosis. As a primary calcium signal transducer, calmodulin (CaM) responds to cytosolic calcium fluxes by binding to and regulating the activity of target CaM-binding proteins (CaMBPs). Ca(2+)-dependent CaMBPs primarily contain domains (CaMBDs) that can be classified into motifs based upon variations on the basic amphiphilic alpha-helix domain involving conserved hydrophobic residues at positions 1-10, 1-14 or 1-16. In contrast, an IQ or IQ-like domain often mediates Ca(2+)-independent CaM-binding. Based on these attributes, a search for CaMBDs reveals that many of the proteins intimately linked to AD may be calmodulin-binding proteins, opening new avenues for research on this devastating disease. PMID:15249195

  9. Distribution of calmodulin in corn seedlings - Immunocytochemical localization in coleoptiles and root apices

    Science.gov (United States)

    Dauwalder, M.; Roux, S. J.

    1986-01-01

    Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in etiolated corn (Zea mays, var. Bear Hybrid) seedlings. Uniform staining was seen in the background cytoplasm of most cell types. Cell walls and vacuoles were not stained. In coleoptile mesophyll cells the nucleoplasm of most nuclei was stained as was the stroma of most amyloplasts. The lumen border of mature tracheary elements in coleoptiles also stained. In the rootcap the most intensely stained regions were the cytoplasms of columella cells and of the outermost cells enmeshed in the layer of secreted slime. Nuclei in the rootcap cells did not stain distinctly, but those in all cell types of the root meristem did. Also in the root meristem, the cytoplasm of metaxylem elements stained brightly. These results are compared and contrasted with previous data on the localization of calmodulin in pea root apices and epicotyls and discussed in relation to current hypotheses on mechanisms of gravitropism.

  10. Cloning and Structural Analysis of Calmodulin Gene from the Mangrove Plant Sonneratia Paracaseolaris

    Institute of Scientific and Technical Information of China (English)

    Xiong Lingyuan; Lin Tao; Zhou Hantao; Xu Jinsen; Ge Yunsheng; Chen Muchuan; Chen Liang

    2002-01-01

    Calmodulin is a calcium binding protein that modulates the activity of diverse groups of protein including some protein kinase, adenylate cyclases and ATPase. Here we use the total DNA of Sonneratiaparacaseolaris as the template ofthe polymerase chain reaction (PCR). The PCR primers have been designed and synthesized according to the 5-and 3-terminal oligonucleotide sequences of Calmodulin gene of plants in Genbank and ligated with cloning vector pBsk(+).The recombinant clones have been obtained from the selected medium. The results of DNA sequences analysis show that the nucleotide sequences of ORF share more than 85% homologies as compared with those ofcalmodulin genes of several other plants. Similar to rice and apple, the ORF is interrupted by an intron behind the 75th nucleotide.

  11. Calmodulin-binding transcription activators and perspectives for applications in biotechnology.

    Science.gov (United States)

    Shen, Chenjia; Yang, Yanjun; Du, Liqun; Wang, Huizhong

    2015-12-01

    In recent years, a novel family of calmodulin-binding transcription activators (CAMTAs) has been reported in various species. The CAMTAs share a conserved domain organization, with a CG-1 DNA-binding domain, a transcription factor immunoglobulin domain, several ankyrin repeats, a calmodulin-binding domain, and a varying number of IQ motifs. CAMTAs participate in transcriptional regulation by recognizing and binding to a specific cis-element: (G/A/C)CGCG(C/G/T). Plants suffer from the environmental challenges, including abiotic and biotic stresses. Investigations in various plant species indicate a broad range of CAMTA functions involved in developmental regulation, environmental stress response, and hormone cross talk. In this review, we focus on the expression patterns and biological functions of CAMTAs to explore their probable applications in biotechnology. Furthermore, the identification and phylogenetic analysis of CAMTAs in crops could open new perspectives for enhancing stress tolerance, which could lead to improved crop production.

  12. Calmodulin-binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis.

    Science.gov (United States)

    Galon, Yael; Nave, Roy; Boyce, Joy M; Nachmias, Dikla; Knight, Marc R; Fromm, Hillel

    2008-03-19

    Calmodulin-binding transcription activator (CAMTA) 3 (also called SR1) is a calmodulin-binding transcription factor in Arabidopsis. Two homozygous T-DNA insertion mutants (camta3-1, camta3-2) showed enhanced spontaneous lesions. Transcriptome analysis of both mutants revealed 6 genes with attenuated expression and 99 genes with elevated expression. Of the latter, 32 genes are related to defense against pathogens (e.g. WRKY33, PR1 and chitinase). Propagation of a virulent strain of the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea were attenuated in both mutants. Moreover, both mutants accumulated high levels of H2O2. We suggest that CAMTA3 regulates the expression of a set of genes involved in biotic defense responses.

  13. Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels

    OpenAIRE

    Meissner, Gerhard; Pasek, Daniel A.; Yamaguchi, Naohiro; Ramachandran, Srinivas; Dokholyan, Nikolay V.; Tripathy, Ashutosh

    2009-01-01

    The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [35S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca2+ concentrations from

  14. Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

    OpenAIRE

    C. Yan; Zhao, A Z; Bentley, J K; Loughney, K; Ferguson, K; Beavo, J. A.

    1995-01-01

    The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-...

  15. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    OpenAIRE

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII e...

  16. Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy

    OpenAIRE

    Li, Hui; Li, Weiwei; Arun K Gupta; Mohler, Peter J.; Anderson, Mark E.; Grumbach, Isabella M.

    2009-01-01

    Despite our understanding that medial smooth muscle hypertrophy is a central feature of vascular remodeling, the molecular pathways underlying this pathology are still not well understood. Work over the past decade has illustrated a potential role for the multifunctional calmodulin-dependent kinase CaMKII in smooth muscle cell contraction, growth, and migration. Here we demonstrate that CaMKII is enriched in vascular smooth muscle (VSM) and that CaMKII inhibition blocks ANG II-dependent VSM c...

  17. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    Science.gov (United States)

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  18. Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize

    Science.gov (United States)

    Stinemetz, C. L.; Hasenstein, K. H.; Young, L. M.; Evans, M. L.

    1992-01-01

    We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 micromole) that did not affect root growth. Calmodulin antagonists (> or = 1 micromole) inhibited downward transport of label from 45Ca2+ across the caps of gravistimulated roots relative to the downward transport of 45Ca2+ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip (> or = 1 micromole) also decreased the relative downward movement of label from 3H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of 45Ca2+ in the root tip nor the upward movement of label from 3H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists > or = 1 micromole become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.

  19. Calmodulin-dependent protein kinases mediate calcium-induced slow motility of mammalian outer hair cells.

    Science.gov (United States)

    Puschner, B; Schacht, J

    1997-08-01

    Cochlear outer hair cells in vitro respond to elevation of intracellular calcium with slow shape changes over seconds to minutes ('slow motility'). This process is blocked by general calmodulin antagonists suggesting the participation of calcium/calmodulin-dependent enzymatic reactions. The present study proposes a mechanism for these reactions. Length changes of outer hair cells isolated from the guinea pig cochlea were induced by exposure to the calcium ionophore ionomycin. ATP levels remained unaffected by this treatment ruling out depletion of ATP (by activation of calcium-dependent ATPases) as a cause of the observed shape changes. Involvement of protein kinases was suggested by the inhibition of shape changes by K252a, a broad-spectrum inhibitor of protein kinase activity. Furthermore, the inhibitors ML-7 and ML-9 blocked the shape changes at concentrations compatible with inhibition of myosin light chain kinase (MLCK). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), also attenuated the length changes. Inhibitors with selectivity for cyclic nucleotide-dependent protein kinases (H-89, staurosporine) were tested to assess potential additional contributions by such enzymes. The dose dependence of their action supported the notion that the most likely mechanism of slow motility involves phosphorylation reactions catalyzed by MLCK or CaMKII or both. PMID:9282907

  20. Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C.

    Science.gov (United States)

    Raufman, J P; Malhotra, R; Xie, Q; Raffaniello, R D

    1997-03-01

    In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells

  1. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Science.gov (United States)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  2. Biosensor-based approach identifies four distinct calmodulin-binding domains in the G protein-coupled estrogen receptor 1.

    Directory of Open Access Journals (Sweden)

    Quang-Kim Tran

    Full Text Available The G protein-coupled estrogen receptor 1 (GPER has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca(2+-elevating agent. To confirm direct interaction and locate the calmodulin-binding domain(s, we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER's submembrane domains (SMDs. Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83-93, 150-175, 242-259, 330-351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca(2+-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent K d values being 0.44 ± 0.03, 1.40 ± 0.16, 8.01 ± 0.29, and 136.62 ± 6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca(2+ indicators identified separate Ca(2+ sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca(2+ response, representing two distinct species (SMD1 sp1 and SMD1 sp2 with drastically different Ca(2+ sensitivities. The Ca(2+ sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca(2+ values being 0.13 ± 0.02, 0.75 ± 0.05, 2.38 ± 0.13, 3.71 ± 0.13, and 5.15 ± 0.25 µM, respectively. These data indicate that calmodulin may regulate GPER

  3. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen;

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2...

  4. Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.

    Science.gov (United States)

    Ozaki, Hana; Katoh, Tsuyoshi; Nakagawa, Ryoko; Ishihara, Yasuhiro; Sueyoshi, Noriyuki; Kameshita, Isamu; Taniguchi, Takanobu; Hirano, Tetsuo; Yamazaki, Takeshi; Ishida, Atsuhiko

    2016-09-01

    Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation. PMID:27369073

  5. Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

    Science.gov (United States)

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

    2014-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. PMID:25064143

  6. Regulation of Voltage-Gated Ca2+ Currents by Ca2+/Calmodulin-dependent Protein Kinase II in Resting Sensory Neurons

    Science.gov (United States)

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H.

    2014-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca2+ channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca2+ currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16 – 30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. PMID:25064143

  7. Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode*

    OpenAIRE

    Ishida, Hiroaki; Rainaldi, Mario; Vogel, Hans J.

    2009-01-01

    The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca2+-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca2+-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of b...

  8. MIPS: a calmodulin-binding protein of Gracilaria lemaneiformis under heat shock.

    Science.gov (United States)

    Zhang, Xuan; Zhou, Huiyue; Zang, Xiaonan; Gong, Le; Sun, Hengyi; Zhang, Xuecheng

    2014-08-01

    To study the Ca(2+)/Calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, myo-inositol-1-phosphate synthase (MIPS), a calmodulin-binding protein, was isolated using the yeast two-hybrid system. cDNA and DNA sequences of mips were cloned from G. lemaneiformis by using 5'RACE and genome walking procedures. The MIPS DNA sequence was 2,067 nucleotides long, containing an open reading frame (ORF) of 1,623 nucleotides with no intron. The mips ORF was predicted to encode 540 amino acids, which included the conserved MIPS domain and was 61-67 % similar to that of other species. After analyzing the amino acid sequence of MIPS, the CaM-Binding Domain (CaMBD) was inferred to be at a site spanning from amino acid 212 to amino acid 236. The yeast two-hybrid results proved that MIPS can interact with CaM and that MIPS is a type of calmodulin-binding protein. Next, the expression of CaM and MIPS in wild-type G. lemaneiformis and a heat-tolerant G. lemaneiformis cultivar, "981," were analyzed using real-time PCR under a heat shock of 32 °C. The expression level displayed a cyclical upward trend. Compared with wild type, the CaM expression levels of cultivar 981 were higher, which might directly relate to its resistance to high temperatures. This paper indicates that MIPS and CaM may play important roles in the high-temperature resistance of G. lemaneiformis.

  9. Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Poovaiah, B. W.

    2002-01-01

    Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.

  10. Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

    Science.gov (United States)

    Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

    1999-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

  11. Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulin-dependent protein kinase homolog

    Science.gov (United States)

    Lu, Y. T.; Feldman, L. J.

    1997-01-01

    Roots of many species grow downward (orthogravitropism) only when illuminated. Previous work suggests that this is a calcium-regulated response and that both calmodulin and calcium/calmodulin-dependent kinases participate in transducing gravity and light stimuli. A genomic sequence has been obtained for a calcium/calmodulin-dependent kinase homolog (MCK1) expressed in root caps, the site of perception for both light and gravity. This homolog consists of 7265 base pairs and contains 11 exons and 10 introns. Since MCK1 is expressed constitutively in both light and dark, it is unlikely that the light directly affects MCK1 expression, though the activity of the protein may be affected by light. In cultivars showing light-regulated gravitropism, we hypothesize that MCK1, or a homolog, functions in establishing the auxin asymmetry necessary for orthogravitropism.

  12. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses

    OpenAIRE

    Heo, Won Do; Lee, Sang Hyoung; Kim, Min Chul; Kim, Jong Cheol; Chung, Woo Sik; Chun, Hyun Jin; Lee, Kyoung Joo; Park, Chan Young; Park, Hyeong Cheol; Choi, Ji Young; Cho, Moo Je

    1999-01-01

    The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, wher...

  13. Role of calcium and calmodulin in reaction of gastric fundus contraction

    Directory of Open Access Journals (Sweden)

    Marta Gajdus

    2011-09-01

    Full Text Available Background:The subject of this study is determination of the influence of calmodulin and calcium on gastric fundus smooth muscle contraction. During experiments, the author tested the influence of a serotonin receptor agonist, serotonin (5-HT, causing smooth muscle contraction.Material/Methods:Testing was conducted on tissues isolated from rat’s stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg. The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification.Results:According to conducted tests, we can deduce that 8 Br cGMP stops the reaction of gastric fundus smooth muscle contraction induced by serotonin. The use of 8Br-cGMP in the range of concentrations between 10 and 300 µM leads to reduction of maximum effect from 100�0to 46�20Similar changes were obtained after the use of guanylate cyclase activator (CG – YC-1. Curves for the contractile activity of serotonin along with an increase of concentration YC-1 are shifted to the right, and the maximum effect of reaction decreases. Increasing concentrations of flunarizine, a calmodulin antagonist, in a concentration-dependent way blocks binding between calcium and calmodulin, and at the same time leads to the shift of concentration-effect curves for serotonin to the right and a decrease of maximum reaction.Increasing concentrations of ODQ, a guanylate cyclase inhibitor lead to statistically significant shift of the curves to the left, decrease of EC50 value and simultaneous increase of maximum reaction to serotonin.Conclusions:According to conducted testing, serotonin causes gastric fundus smooth muscle contraction dependent on

  14. Postsynaptic long-term enhancement (LTE) by dopamine may be mediated by Ca2+ and calmodulin.

    Science.gov (United States)

    Mochida, S; Libet, B

    1990-04-01

    Long-term enhancement (LTE), of postsynaptic slow depolarizing responses to a muscarinic agonist (MCh), follows a brief exposure of the rabbit superior cervical ganglion to another transmitter, dopamine (DA). Either reduction of external Ca2+ (to 1.0 mM or 0.2 mM) or presence of a specific calmodulin antagonist (calmidazolium at 5 microM) blocked DA induction of this LTE. However, unlike LTP in hippocampus, induction of LTE is not mediated by depolarization-dependent influx of Ca2+.

  15. An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility.

    Science.gov (United States)

    Suarez, Andres; Huber, Robert J; Myre, Michael A; O'Day, Danton H

    2011-07-01

    CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote. PMID:21402150

  16. Munc13-like skMLCK variants cannot mimic the unique calmodulin binding mode of Munc13 as evidenced by chemical cross-linking and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Sabine Herbst

    Full Text Available Among the neuronal binding partners of calmodulin (CaM are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif's core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca(2+ concentrations.

  17. Retinoic acid inhibits calmodulin binding to human erythrocyte membranes and reduces membrane Ca2(+)-adenosine triphosphatase activity.

    OpenAIRE

    Davis, F B; Smith, T. J.; Deziel, M R; Davis, P J; Blas, S D

    1990-01-01

    Ca2(+)-ATPase activity in human red cell membranes is dependent on the presence of calmodulin. All trans-retinoic acid inhibited human red cell membrane Ca2(+)-ATPase activity in vitro in a concentration-dependent manner (10(-8) to 10(-4) M). In contrast, retinol, retinal, 13-cis-retinoic acid and the benzene ring analogue of retinoic acid did not alter enzyme activity. Purified calmodulin (up to 500 ng/ml, 3 X 10(-8) M) added to red cell membranes, in the presence of inhibitory concentration...

  18. Protective effects of calmodulin antagonists (trifluoperazine and W-7 on hypothermic ischemic rat hearts.

    Directory of Open Access Journals (Sweden)

    Sugawara,Eiji

    1991-06-01

    Full Text Available The cardioprotective effect of calmodulin antagonists, trifluoperazine (TFP and N-(6-aminohexyl-5-chloro-1-naphthalene sulfonamide (W-7 was examined on the isolated rat heart exposed to hypothermic and ischemic conditions by measuring distribution of lysosomal enzymes in myocardial cells, and leakage of creatine kinase (CK during reperfusion and postischemic recovery in myocardial systolic function. Experimental hearts were infused with 20 degrees C Krebs-Henseleit bicarbonate buffer (KHB or KHB containing TFP or W-7 for 2min every 30min during hypothermic ischemia. After ischemia for 120min at 20 degrees C, rat hearts were reperfused at 37 degrees C for 30min. TFP and W-7 improved functional recovery and prevented CK release. In TFP treated hearts, leakage of lysosomal enzymes was reduced significantly, whereas stabilization of lysosomes by W-7 did not occur. These results suggest that calcium-calmodulin dependent enzymes may play an important role in the development of cellular damage of the myocardium during hypothermic ischemia, although levels of leakage of lysosomal enzymes may be unreliable predictors of functional recovery after hypothermic ischemia.

  19. Resveratrol increases nitric oxide production in the rat thick ascending limb via Ca2+/calmodulin.

    Science.gov (United States)

    Gonzalez-Vicente, Agustin; Cabral, Pablo D; Garvin, Jeffrey L

    2014-01-01

    The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca2+ (Cai) and AMP kinase (AMPK) and NAD-dependent deacetylase sirtuin1 (SIRT1) activities, all of which could activate NO production. We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca2+/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Cai was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (pthick ascending limbs via a Ca2+/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects.

  20. NMR and molecular dynamics studies of the interaction of melatonin with calmodulin

    Science.gov (United States)

    Turjanski, Adrián G.; Estrin, Darío A.; Rosenstein, Ruth E.; McCormick, John E.; Martin, Stephen R.; Pastore, Annalisa; Biekofsky, Rodolfo R.; Martorana, Vincenzo

    2004-01-01

    Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca2+ concentration via activation of its G-protein–coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring 15N and 1H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance. PMID:15498938

  1. Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

    Science.gov (United States)

    Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Giorgetti, Alejandro; Dominici, Paola; Astegno, Alessandra

    2016-08-01

    In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. PMID:27124620

  2. Isolation of Hybridomas for Golgi-associated Proteins and a Plant Calmodulin

    Science.gov (United States)

    Kuzmanoff, K. M.; Ray, P. M.

    1985-01-01

    The demonstration of a role for calcium in the mechanism of the gravitropic response indicates a role for calmodulin. Localization studies indicate that plant cell walls have a high content of calmodulin which suggests a regulatory role for CaM in both gravitropic curvature and auxin-induced growth. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the ctivity of Golgi-localized B-1,4-glucan synthase (GS), an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. In order to determine whether auxin stimulates GS activity either by modulation of existing enzyme or induces de novo formation of Golgi glucan synthase, a study was undertaken to isolate and quantitate glucan synthase. This enzyme appears to be an integral protein of the Golgi membrane and has resisted isolation with retention of activity. The production of monoclonal antibody for glucan synthase was undertaken due to the inability to isolate GS by standard detergent/liposome techniques.

  3. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  4. SPLICE VARIANT SPECIFIC UPREGULATIONOF CA+2/CALMODULIN DEPENDENT PROTEIN KINASE 1G BY PYRETHROID INSECTICIDES IN VIVO.

    Science.gov (United States)

    Pyrethroid insecticides induce neurotoxicity in mammals by interfering with ion channel function in excitable neuronal membranes. Previous work demonstrated dose-dependent increases in expression of Ca+2/calmodulin dependent protein kinase (Camk1g) mRNA following acute deltameth...

  5. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  6. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

    Full Text Available This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

  7. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  8. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  9. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  10. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  11. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  12. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    Science.gov (United States)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  13. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    Science.gov (United States)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  14. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  15. Immunohistochemical locali- zation of Ca2+/calmodulin- dependent kinase in tobacco

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The existence of Ca2+/calmodulin-dependent kinase (CaM kinase, CaMK) in tobacco is verified immuno- logically and its distribution in different tissues of tobacco is studied. It has been demonstrated that CaMK is mainly distributed in early developing anthers, developing ovules and embryos, lateral root primordium, apical meristem and leaf primordium of buds and mesophyll cells and developing vascular bundles of leaves. There is enormous CaM kinase distributed in leaf epidermis fair cells and guard cells of stomas too. Little kinase is found in mature stem or root cells. The distribution properties of CaM kinase in tobacco are consistent with those of CaM, suggesting that there exists the Ca2+ signal transduction pathway mediated by CaM kinase in tobacco and it plays an important role in the plant growth and development.

  16. The Ca(2+)/Calmodulin/CaMKK2 Axis: Nature's Metabolic CaMshaft.

    Science.gov (United States)

    Marcelo, Kathrina L; Means, Anthony R; York, Brian

    2016-10-01

    Calcium (Ca(2+)) is an essential ligand that binds its primary intracellular receptor calmodulin (CaM) to trigger a variety of downstream processes and pathways. Central to the actions of Ca(2+)/CaM is the activation of a highly conserved Ca(2+)/CaM kinase (CaMK) cascade that amplifies Ca(2+) signals through a series of subsequent phosphorylation events. Proper regulation of Ca(2+) flux is necessary for whole-body metabolism and disruption of Ca(2+) homeostasis has been linked to various metabolic diseases. Here we provide a synthesis of recent advances that highlight the roles of the Ca(2+)/CaMK axis in key metabolic tissues. An appreciation of this information is critical to understanding the mechanisms by which Ca(2+)/CaM-dependent signaling contributes to metabolic homeostasis and disease.

  17. Genotyping species of the Sporothrix schenckii complex by PCR-RFLP of calmodulin.

    Science.gov (United States)

    Rodrigues, Anderson Messias; de Hoog, G Sybren; de Camargo, Zoilo Pires

    2014-04-01

    Sporotrichosis is one of the most common subcutaneous mycosis in Latin America and is caused by 4 pathogenic thermodimorphic fungi in the genus Sporothrix. From both therapeutic and epidemiological perspectives, it is essential to identify the causative agents down to the species level. Traditional parameters may overlap among closely related species, and we propose polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as an alternative approach. In the present study, the calmodulin gene was amplified and digested with HhaI to yield 5 different electrophoretic patterns representing all medically important Sporothrix species: Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, and Sporothrix luriei. The PCR-RFLP protocol described here is a simple and inexpensive method and is highly suitable for accurate routine genotyping of relevant Sporothrix species.

  18. Assembly and Calcium Binding Properties of Quantum Dot-Calmodulin Calcium Sensor.

    Science.gov (United States)

    Eun, Su-yong; Nguyen-ta, Kim; Yoo, Hoon; Silva, Gabriel A; Kim, Soon-jong

    2016-02-01

    We have developed the first nanoengineered quantum dot molecular complex designed to measure changes of calcium ion (Ca2+) concentration at high spatial and temporal resolutions in real time. The sensor is ratiometric and composed of three components: a quantum dot (QD) emitting at 620 nm as a fluorescence donor, an organic dye (Alexa Fluor 647) as a fluorescence acceptor, and a calmodulin-M13 (CaM-M13) protein part as a calcium sensing component. In this work, we have determined the maximal number of CaM-M13 required for saturating a single QD particle to be approximately 16. The dissociation constant, Kd of the QD-based calcium ion sensor was also estimated to be around 30 microM. PMID:27433729

  19. ARRHYTHMOGENIC CALMODULIN MUTATIONS AFFECT THE ACTIVATION AND TERMINATION OF CARDIAC RYANODINE RECEPTOR MEDIATED CA2+ RELEASE

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Chazin, Walter J.; Chen, Wayne S.R.;

    We recently identified the first two human missense mutations in a calmodulin (CaM) gene (CALM1) and linked these to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death in young individuals1. More CaM mutations have since been identified in CALM1 and also...... in the other two CaM genes (CALM2 and CALM3). All CaM mutations are associated with severe ventricular arrhythmias. CaM regulates several key proteins governing cardiac excitation-contraction coupling (ECC), including the cardiac ryanodine receptor (RyR2) Ca2+ release channel. RyR2 mutations also dominantly...... cause CPVT, where the mutations increase the channel sensitivity to activation and enhance the propensity for pro-arrhythmogenic spontaneous Ca2+ release. Here we investigated the effect of CPVT-linked CaM mutations (N53I and N97S) and two CaM mutations identified in individuals with early onset severe...

  20. Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants

    Institute of Scientific and Technical Information of China (English)

    Min Chul Kim; Woo Sik Chung; Dae-Jin Yun; Moo Je Cho

    2009-01-01

    Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca2+ ion. Calmodulin (CAM) is the predominant Ca2+ sensor and plays a crucial role in decoding the Ca2+ signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca2+ and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca2+ and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues.

  1. Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

    Science.gov (United States)

    Selwa, Edithe; Davi, Marilyne; Chenal, Alexandre; Sotomayor-Pérez, Ana-Cristina; Ladant, Daniel; Malliavin, Thérèse E

    2014-07-25

    Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

  2. Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

    International Nuclear Information System (INIS)

    Highlights: → Both Ca++-Calmodulin (CaM) and Ca++-free CaM bind to the C-terminal region of Nav1.1. → Ca++ and CaM have both opposite and convergent effects on INav1.1. → Ca++-CaM modulates INav1.1 amplitude. → CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. → Ca++ alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca++ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca++ could bind the Nav1.1 C-terminal region with micromolar affinity.

  3. C-terminal extension of calmodulin-like 3 protein from Oryza sativa L.: interaction with a high mobility group target protein.

    Science.gov (United States)

    Chinpongpanich, Aumnart; Phean-O-Pas, Srivilai; Thongchuang, Mayura; Qu, Li-Jia; Buaboocha, Teerapong

    2015-11-01

    A large number of calmodulin-like (CML) proteins are present in plants, but there is little detailed information on the functions of these proteins in rice (Oryza sativa L.). Here, the CML3 protein from rice (OsCML3) and its truncated form lacking the C-terminal extension (OsCML3m) were found to exhibit a Ca2+-binding property and subsequent conformational change, but the ability to bind the CaM kinase II peptide was only observed for OsCML3m. Changes in their secondary structure upon Ca2+-binding measured by circular dichroism revealed that OsCML3m had a higher helical content than OsCML3. Moreover, OsCML3 was mainly localized in the plasma membrane, whereas OsCML3m was found in the nucleus. The rice high mobility group B1 (OsHMGB1) protein was identified as one of the putative OsCML3 target proteins. Bimolecular fluorescence complementation analysis revealed that OsHMGB1 bound OsCML3, OsCML3m or OsCML3s (cysteine to serine mutation at the prenylation site) in the nucleus presumably through the methionine and phenylalanine-rich hydrophobic patches, confirming that OsHMGB1 is a target protein in planta. The effect of OsCML3 or OsCML3m on the DNA-binding ability of OsHMGB1 was measured using an electrophoretic mobility shift assay. OsCML3m decreased the level of OsHMGB1 binding to pUC19 double-stranded DNA whereas OsCML3 did not. Taken together, OsCML3 probably provides a mechanism for manipulating the DNA-binding ability of OsHMGB1 in the nucleus and its C-terminal extension provides an intracellular Ca2+ regulatory switch.

  4. The roles and relations of calpastatin, calmodulin and an undefined cytoplasmic factor in the regulation of cardiac L-type Ca2+ channels

    Institute of Scientific and Technical Information of China (English)

    HAO Li-ying; ZHU Tong; HU Hui-yuan; ZHAO Mei-mi; RUI Feng; LIU Yan; ZHAO Jin-sheng; tsuko Minobe; Masaki Kameyama

    2008-01-01

    Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down'. The factors include ATP, calpastatin and H fraction (a high molecular fraction of bovine cardiac cytoplasm). Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes. Run-down was induced by the inside-out patch formation. Calpastatin (CS), calmodulin(CaM) and three GST-fusion fragment peptides derived from the C-terminal tail of guineapig Car1.2, CT-1 (amino acids number 1509-1791), CTo2 (1777-2003) and CT-3 (1944-2169) were produced as GST fusion proteins. Results (1)CaM + ATP or CS + ATP restored the channels after rundown;however, the CaM or CS's effects became smaller with the longer run-down time. (2)After run down, CaM-dependent protein kinase (CaMKII) produced Ca2+ channel activity to only 2-10% of the basal activity, however, in the presence of CaMKII, the time-dependent nature of the CaM effect was abolished. (3) In pull-down assay, CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase. (4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm. Conclusions The results show that CS, CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels, and that there might be cross talks among the four factors (CS, CaM, CaMKII and the undefined cytoplasmic factor). This work was supported by the grants from the Japan Society for the Promotion of Science and the National Natural Science Foundation of China (No. 30670761, No. 30671726).

  5. Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response through calmodulin-binding transcription factor AtSR1/CAMTA3.

    Science.gov (United States)

    Qiu, Yongjian; Xi, Jing; Du, Liqun; Suttle, Jeffrey C; Poovaiah, B W

    2012-05-01

    Calcium/calmodulin (Ca(2+)/CaM) has long been considered a crucial component in wound signaling pathway. However, very few Ca(2+)/CaM-binding proteins have been identified which regulate plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca(2+)/CaM-binding transcription factors designated as AtSRs (also known as AtCAMTAs) can respond differentially to wounding stress. Further studies revealed that AtSR1/CAMTA3 is a negative regulator of plant defense, and Ca(2+)/CaM-binding to AtSR1 is indispensable for the suppression of salicylic acid (SA) accumulation and disease resistance. Here we report that Ca(2+)/CaM-binding is also critical for AtSR1-mediated herbivore-induced wound response. Interestingly, atsr1 mutant plants are more susceptible to herbivore attack than wild-type plants. Complementation of atsr1 mutant plants by overexpressing wild-type AtSR1 protein can effectively restore plant resistance to herbivore attack. However, when mutants of AtSR1 with impaired CaM-binding ability were overexpressed in atsr1 mutant plants, plant resistance to herbivore attack was not restored, suggesting a key role for Ca(2+)/CaM-binding in wound signaling. Furthermore, it was observed that elevated SA levels in atsr1 mutant plants have a negative impact on both basal and induced biosynthesis of jasmonates (JA). These results revealed that Ca(2+)/CaM-mediated signaling regulates plant response to herbivore attack/wounding by modulating the SA-JA crosstalk through AtSR1. PMID:22371088

  6. Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.

    Science.gov (United States)

    Ordaz, Benito; Tang, Jisen; Xiao, Rui; Salgado, Alfonso; Sampieri, Alicia; Zhu, Michael X; Vaca, Luis

    2005-09-01

    TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

  7. Plant signalling peptides

    OpenAIRE

    Wiśniewska, Justyna; Trejgell, Alina; Tretyn, Andrzej

    2003-01-01

    Biochemical and genetic studies have identified peptides that play crucial roles in plant growth and development, including defence mechanisms in response to wounding by pests, the control of cell division and expansion, and pollen self-incompatibility. The first two signalling peptides to be described in plants were tomato systemin and phytosulfokine (PSK). There is also biochemical evidence that natriuretic peptide-like molecules, immunologically-relatedt o those found ...

  8. Polycyclic peptide therapeutics.

    Science.gov (United States)

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. PMID:23355488

  9. [The structure and phosphorus or potassium deficiency induced expression of a calmodulin-like protein gene in Arabidopsis].

    Science.gov (United States)

    Duan, Rui-Jun; Yi, Ke-Ke; Wu, Ping

    2005-10-01

    According to our previous microarray analysis, we found a putative calmodulin gene related to Pi deficiency and designated AtPsiCaM (Arabidopsis Pi-starvation-induced CaM). Results of structural analysis indicate that AtPsiCaM has three conserved EF-hands motif and belongs to calmodulin-like proteins family (Figs. 1-3). Northern blot analysis revealed that this gene could be induced by potassium and phosphate deficiency and not by potassium deficiency or high salinity (Fig. 4). The results of RT-PCR and GUS histochemical staining assays of the AtPsiCaM promoter::GUS transgenic plants showed that this gene can be expressed in all tissues to different expression levels (Figs. 5, 6). PMID:16222095

  10. Characterization of a calcium/calmodulin-regulated SR/CAMTA gene family during tomato fruit development and ripening

    Directory of Open Access Journals (Sweden)

    Yang Tianbao

    2012-02-01

    Full Text Available Abstract Background Fruit ripening is a complicated development process affected by a variety of external and internal cues. It is well established that calcium treatment delays fruit ripening and senescence. However, the underlying molecular mechanisms remain unclear. Results Previous studies have shown that calcium/calmodulin-regulated SR/CAMTAs are important for modulation of disease resistance, cold sensitivity and wounding response in vegetative tissues. To study the possible roles of this gene family in fruit development and ripening, we cloned seven SR/CAMTAs, designated as SlSRs, from tomato, a model fruit-bearing crop. All seven genes encode polypeptides with a conserved DNA-binding domain and a calmodulin-binding site. Calmodulin specifically binds to the putative targeting site in a calcium-dependent manner. All SlSRs were highly yet differentially expressed during fruit development and ripening. Most notably, the expression of SlSR2 was scarcely detected at the mature green and breaker stages, two critical stages of fruit development and ripening; and SlSR3L and SlSR4 were expressed exclusively in fruit tissues. During the developmental span from 10 to 50 days post anthesis, the expression profiles of all seven SlSRs were dramatically altered in ripening mutant rin compared with wildtype fruit. By contrast, only minor alterations were noted for ripening mutant nor and Nr fruit. In addition, ethylene treatment of mature green wildtype fruit transiently stimulated expression of all SlSRs within one to two hours. Conclusions This study indicates that SlSR expression is influenced by both the Rin-mediated developmental network and ethylene signaling. The results suggest that calcium signaling is involved in the regulation of fruit development and ripening through calcium/calmodulin/SlSR interactions.

  11. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

    OpenAIRE

    Glaser, P; Elmaoglou-Lazaridou, A; Krin, E.; Ladant, D.; Bârzu, O; Danchin, A

    1989-01-01

    In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture sup...

  12. Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

    DEFF Research Database (Denmark)

    Shi, Juan; Geshi, Naomi; Takahashi, Shinichi;

    2013-01-01

    The molecular mechanism underlying Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cel...... essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells....

  13. Dendritic spine changes in the development of alcohol addiction regulated by α-calcium/calmodulin-dependent protein kinase II

    OpenAIRE

    Zofia Mijakowska

    2014-01-01

    Introduction Alcohol has many adverse effects on the brain. Among them are dendritic spine morphology alterations, which are believed to be the basis of alcohol addiction. Autophosphorylation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII) has been shown to regulate spine morphology in vitro. Here we show that αCaMKII can also regulate addiction related behaviour and dendritic spine morphology changes caused by alcohol consumption in vivo. Method 12 αCaMKII-autophosphorylatio...

  14. Insulin C-peptide test

    Science.gov (United States)

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  15. The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast.

    Science.gov (United States)

    Craig, R; Norbury, C

    1998-12-18

    Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes. PMID:9819352

  16. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  17. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    Science.gov (United States)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  18. Comparing allosteric transitions in the domains of calmodulin through coarse-grained simulations

    CERN Document Server

    Nandigrami, Prithviraj

    2015-01-01

    Calmodulin (CaM) is a ubiquitous calcium binding protein consisting of two structurally similar domains with distinct stabilities, binding affinities, and flexibilities. We present coarse grained simulations that suggest the mechanism for the domain's allosteric transitions between the open and closed conformations depend on subtle differences in the folded state topology of the two domains. Throughout a wide temperature range, the simulated transition mechanism of the N-terminal domain (nCaM) follows a two-state transition mechanism while domain opening in the C-terminal domain (cCaM) involves unfolding and refolding of the tertiary structure. The appearance of the unfolded intermediate occurs at a higher temperature in nCaM than it does in cCaM. That is, we find that cCaM unfolds more readily along the transition route than nCaM. Furthermore, unfolding and refolding of the domain significantly slows the domain opening and closing rates of cCaM, a distinct scenario which can potentially influence the mechani...

  19. High-level expression of human calmodulin in E. coli and its effects on cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Xiao Jun Li; Jian Guo Wu; Jun Ling Si; Da Wen Guo; Jian Ping Xu

    2000-01-01

    Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1,2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.

  20. Growth, Gas Exchange, Abscisic Acid, and Calmodulin Response to Salt Stress in Three Poplars

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present study, we investigated the effects of increasing salinity on growth, gas exchange, abscisic acid(ABA), calmodulin (CAM), and the relevance to salt tolerance in seedlings of Populus euphratica Oliv. and cuttings of P. "pupularis 35-44" (P. popularis) and P. x euramericana cv. 1-214 (P. cv. Italica). The relative growth rates of shoot height (RGRH) for P. cv. Italica and P. popularis were severely reduced by increasing salt stress,whereas the growth reduction was relatively less in P. euphratica. Similarly, P. euphratica maintained higher net photosynthetic rates (Pn) and unit transpiration rates (TRN) than P. cv. Italica and P. popularis under conditions of higher salinity. Salinity caused a significant increase in leaf ABA and CaM in the three genotypes after the onset of stress, but NaCl-induced ABA and CaM accumulation was more pronounced in P. euphratica,suggesting that P. euphratica plants are more sensitive in sensing soil salinity than the other two poplars.Furthermore, P. euphratica maintained relatively higher ABA and CaM concentrations under conditions of high salinity. The higher capacity to synthesize stress signals, namely ABA and CaM, in P. euphratica and the contribution of this to the salt resistance of P. euphratica are discussed.

  1. Calmodulin Involvement in Stress-Activated Nuclear Localization of Albumin in JB6 Epithelial Cells.

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Thomas J.; Negash, Sewite; Smallwood, Heather S.; Ramos, Kenneth S.; Thrall, Brian D.; Squier, Thomas C.

    2004-06-15

    We report that in response to oxidative stress, albumin is translocated to the nucleus where it binds in concert with known transcription factors to an antioxidant response element (ARE), which controls the expression of glutathione-S-transferase and other antioxidant enzymes, functioning to mediate adaptive cellular responses. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein, calmodulin (CaM), co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients, and associated increases in reactive oxygen species, contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin to mediate the transcriptional regulation of antioxidant responsive elements.

  2. Distinguishing Unfolding and Functional Conformational Transitions of Calmodulin Using Ultraviolet Resonance Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Eric M.; Balakrishnan, G.; Squier, Thomas C.; Spiro, Thomas

    2014-06-14

    Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium-binding protein exhibits a broad range of structural transitions, including domain opening and folding-unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well known, it has been challenging to examine the dynamics of transition between these structures in real-time, due to a lack of suitable spectroscopic probes of CaM structure. In this paper, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C-terminal domain in response to Ca2+ binding. This spectral difference is entirely due to differences in teritary contacts at the inter-domain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation-sensitive tyrosine residues, opening a path to time-resolved studies of CaM dynamics using Raman spectroscopy.

  3. The Ca(2+ influence on calmodulin unfolding pathway: a steered molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    Full Text Available The force-induced unfolding of calmodulin (CaM was investigated at atomistic details with steered molecular dynamics. The two isolated CaM domains as well as the full-length CaM were simulated in N-C-terminal pulling scheme, and the isolated N-lobe of CaM was studied specially in two other pulling schemes to test the effect of pulling direction and compare with relevant experiments. Both Ca(2+-loaded CaM and Ca(2+-free CaM were considered in order to define the Ca(2+ influence to the CaM unfolding. The results reveal that the Ca(2+ significantly affects the stability and unfolding behaviors of both the isolated CaM domains and the full-length CaM. In Ca(2+-loaded CaM, N-terminal domain unfolds in priori to the C-terminal domain. But in Ca(2+-free CaM, the unfolding order changes, and C-terminal domain unfolds first. The force-extension curves of CaM unfolding indicate that the major unfolding barrier comes from conquering the interaction of two EF-hand motifs in both N- and C- terminal domains. Our results provide the atomistic-level insights in the force-induced CaM unfolding and explain the observation in recent AFM experiments.

  4. Calmodulin activation by calcium transients in the postsynaptic density of dendritic spines.

    Directory of Open Access Journals (Sweden)

    Daniel X Keller

    Full Text Available The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.

  5. Calmodulin effects on steroids-regulated plasma membrane calcium pump activity.

    Science.gov (United States)

    Zylinska, Ludmila; Kowalska, Iwona; Ferenc, Bozena

    2009-03-01

    It is now generally accepted that non-genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca(2+) is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca(2+) increase is ATP-consuming plasma membrane calcium pump (PMCA). The enzyme is coded by four genes, but isoforms diversity was detected in excitable and non-excitable cells. It is the only ion pump stimulated directly by calmodulin (CaM). We examined the role of PMCA isoforms composition and CaM effect in regulation of Ca(2+) uptake by estradiol, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and their sulfates in a concentration range from 10(-9) to 10(-6) M, using the membranes from rat cortical synaptosomes, differentiated PC12 cells, and human erythrocytes. In excitable membranes with full set of PMCAs steroids apparently increased Ca(2+) uptake, although to a variable extent. In most of the cases, CaM decreased transport by 30-40% below controls. Erythrocyte PMCA was regulated by the steroids somewhat differently than excitable cells. CaM strongly increased the potency for Ca(2+) extrusion in membranes incubated with 17-beta-estradiol and PREG. Our results indicated that steroids may sufficiently control cytoplasmic calcium concentration within physiological and therapeutic range. The response depended on the cell type, PMCA isoforms expression profile, CaM presence, and the steroids structure. PMID:19226536

  6. Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans

    KAUST Repository

    Chen, Zhangfan

    2012-08-01

    The polychaete . Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of . H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of . H. elegans. A full-length . CaM cDNA was successfully cloned from . H. elegans (. He-CaM) and it contained an open reading frame of 450. bp, encoding 149 amino acid residues. It was highly expressed in 12. h post-metamorphic juveniles, and remained high in adults. . In situ hybridization conducted in competent larvae and juveniles revealed that . He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that . He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of . H. elegans. © 2012 Elsevier Inc..

  7. Calcium/calmodulin-dependent protein kinase IV: A multifunctional enzyme and potential therapeutic target.

    Science.gov (United States)

    Naz, Huma; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-05-01

    The calcium/calmodulin-dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family, and is primarily involved in transcriptional regulation in lymphocytes, neurons and male germ cells. CAMKIV operates the signaling cascade and regulates activity of several transcription activators by phosphorylation, which in turn plays pivotal roles in immune response, inflammation and memory consolidation. In this review, we tried to focus on different aspects of CAMKIV to understand the significance of this protein in the biological system. This enzyme is associated with varieties of disorders such as cerebral hypoxia, azoospermia, endometrial and ovarian cancer, systemic lupus, etc., and hence it is considered as a potential therapeutic target. Structure of CAMKIV is comprised of five distinct domains in which kinase domain is responsible for enzyme activity. CAMKIV is involved in varieties of cellular functions such as regulation of gene expression, T-cell maturation, regulation of survival phase of dendritic cells, bone growth and metabolism, memory consolidation, sperm motility, regulation of microtubule dynamics, cell-cycle progression and apoptosis. In this review, we performed an extensive analysis on structure, function and regulation of CAMKIV and associated diseases. PMID:26773169

  8. Subtle pH differences trigger single residue motions for moderating conformations of calmodulin

    CERN Document Server

    Atilgan, Ali Rana; Atilgan, Canan

    2011-01-01

    This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca2+ signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations to analyze conformational preferences of calcium-loaded CaM, initially in extended form. PRS is comprised of sequential application of directed forces on residues followed by recording the resulting coordinates. We show that manipulation of a single residue, E31 located in one of the EF hand motifs, reproduces structural changes to compact forms, and the flexible linker acts as a transducer of binding information to distant parts of the protein. Independently, using four different pKa calculation strategies, we find E31...

  9. Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation.

    Science.gov (United States)

    Reichow, Steve L; Gonen, Tamir

    2008-09-10

    Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation. PMID:18786401

  10. Comprehensive behavioral analysis of calcium/calmodulin-dependent protein kinase IV knockout mice.

    Directory of Open Access Journals (Sweden)

    Keizo Takao

    Full Text Available Calcium-calmodulin dependent protein kinase IV (CaMKIV is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency.

  11. Mediation by calcium/calmodulin-dependent protein kinase Ⅱ of suppression of GABAA receptors by NMDA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on -aminobutyric acid (GABA)γ-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and IMus), respectively in the Mg2+-free external solution containing 1 mol/L glycine at a holding potential (VH) of 40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 mol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 mol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 mol/L) or La3+ (30 mol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.

  12. Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on -aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (I) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and Imus), respectively in the Mg2+-free external solution containing 1 mol/L glycine at a holding potential (VH) of 40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 mol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 mol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 mol/L) or La3+ (30 mol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.

  13. Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release.

    Science.gov (United States)

    Taylor, James E; Chow, John Y H; Jeffries, Cy M; Kwan, Ann H; Duff, Anthony P; Hamilton, William A; Trewhella, Jill

    2012-08-01

    Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ~70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca(2+) or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca(2+)-signaling during viral processing. PMID:22947870

  14. Cllmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines.

    Science.gov (United States)

    Haußer, I; Herth, W; Reiss, H D

    1984-09-01

    Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming. PMID:24253945

  15. Progress in the participation of Ca2+-calmodulin in heat shock signal transduction

    Institute of Scientific and Technical Information of China (English)

    Rengang Zhou; Bing Li; Hongtao Liu; Daye Sun

    2009-01-01

    A novel heat shock (HS) signal transduction pathway in plants for the participation of Ca2+-calmodulin (CAM) in HS signal trans-duction was identified. HS induces a rapid increase in intracellular free calcium ion levels ([Ca2+]i), and the involvement of phospholipase C-inositol 1,4,5-trisphosphate is one of the factors leading to elevation in [Ca2+]i induced by HS. HS also increases the expression of the CaM gene and the accumulation of the CaM protein. The CaM isoform, AtCaM3, in Arabidopsis is a key member in the HS signal trans-duction pathway. AtCaM3 regulates the activity of CaM-binding protein kinase (AtCBK3) or protein phosphatase (AtPP7), promoting the activation of the HS transcription factor, AtHSFA1a, by phosphorylation/dephosphorylation and the expression of heat shock pro-tein genes, then improving heat tolerance in plants.

  16. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    Directory of Open Access Journals (Sweden)

    Grant S. Nichols

    2015-01-01

    Full Text Available Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX, the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults.

  17. Determination of nutritional and bioactive properties of peptides in enzymatic pea, chickpea, and mung bean protein hydrolysates.

    Science.gov (United States)

    Aluko, Rotimi E

    2008-01-01

    Within the primary structure of many pea and mung bean proteins are peptide sequences that can potentially be used in the formulation of therapeutic products for the treatment and prevention of human diseases. However, these peptide sequences need protease treatments before they can be released free of the parent proteins. Unlike chemical hydrolysis, enzymatic treatment enables more efficient tailoring of peptide products without formation of toxic by-products or destruction of amino acids. This review provides information on current methods that have been used to convert inactive pea and mung bean proteins into bioactive peptides. It focuses on 3 main bioactive properties, such as inhibitions of (1) angiotensin converting enzyme (ACE) activity; (2) calmodulin (CaM)-dependent enzymes; and (3) copper-chelating activity. ACE is an established marker for hypertension, high levels of some CaM-dependent enzymes are risk factors for various human diseases including cancer and Alzheimer's disease, and high vascular copper concentrations may potentiate atherosclerosis. Also reviewed are the production and evaluation of activity of hypoallergenic peptides that may offer protection against anaphylactic reactions. The 3 main proteins discussed are chickpea, mung bean, and field pea.

  18. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.;

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  19. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  20. Introduction to Peptide Synthesis

    OpenAIRE

    Stawikowski, Maciej; Fields, Gregg B.

    2002-01-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  1. Regulation of K-Ras4B Membrane Binding by Calmodulin.

    Science.gov (United States)

    Sperlich, Benjamin; Kapoor, Shobhna; Waldmann, Herbert; Winter, Roland; Weise, Katrin

    2016-07-12

    K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell. PMID:27410739

  2. Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

    Science.gov (United States)

    Oo, Ye Win; Gomez-Hurtado, Nieves; Walweel, Kafa; van Helden, Dirk F; Imtiaz, Mohammad S; Knollmann, Bjorn C; Laver, Derek R

    2015-07-01

    Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed. PMID:25920678

  3. Designing molecular dynamics simulations to shift populations of the conformational states of calmodulin.

    Directory of Open Access Journals (Sweden)

    Ayse Ozlem Aykut

    Full Text Available We elucidate the mechanisms that lead to population shifts in the conformational states of calcium-loaded calmodulin (Ca(2+-CaM. We design extensive molecular dynamics simulations to classify the effects that are responsible for adopting occupied conformations available in the ensemble of NMR structures. Electrostatic interactions amongst the different regions of the protein and with its vicinal water are herein mediated by lowering the ionic strength or the pH. Amino acid E31, which is one of the few charged residues whose ionization state is highly sensitive to pH differences in the physiological range, proves to be distinctive in its control of population shifts. E31A mutation at low ionic strength results in a distinct change from an extended to a compact Ca(2+-CaM conformation within tens of nanoseconds, that otherwise occur on the time scales of microseconds. The kinked linker found in this particular compact form is observed in many of the target-bound forms of Ca(2+-CaM, increasing the binding affinity. This mutation is unique in controlling C-lobe dynamics by affecting the fluctuations between the EF-hand motif helices. We also monitor the effect of the ionic strength on the conformational multiplicity of Ca(2+-CaM. By lowering the ionic strength, the tendency of nonspecific anions in water to accumulate near the protein surface increases, especially in the vicinity of the linker. The change in the distribution of ions in the vicinal layer of water allows N- and C- lobes to span a wide variety of relative orientations that are otherwise not observed at physiological ionic strength. E31 protonation restores the conformations associated with physiological environmental conditions even at low ionic strength.

  4. Localization and function of calmodulin in live-cells of Aspergillus nidulans.

    Science.gov (United States)

    Chen, Shaochun; Song, Yiju; Cao, Jinling; Wang, Gang; Wei, Hua; Xu, Xushi; Lu, Ling

    2010-03-01

    Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca(2+). Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP-CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP-CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP-CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkörper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi.

  5. α-Calcium calmodulin kinase II modulates the temporal structure of hippocampal bursting patterns.

    Directory of Open Access Journals (Sweden)

    Jeiwon Cho

    Full Text Available The alpha calcium calmodulin kinase II (α-CaMKII is known to play a key role in CA1/CA3 synaptic plasticity, hippocampal place cell stability and spatial learning. Additionally, there is evidence from hippocampal electrophysiological slice studies that this kinase has a role in regulating ion channels that control neuronal excitability. Here, we report in vivo single unit studies, with α-CaMKII mutant mice, in which threonine 305 was replaced with an aspartate (α-CaMKII(T305D mutants, that indicate that this kinase modulates spike patterns in hippocampal pyramidal neurons. Previous studies showed that α-CaMKII(T305D mutants have abnormalities in both hippocampal LTP and hippocampal-dependent learning. We found that besides decreased place cell stability, which could be caused by their LTP impairments, the hippocampal CA1 spike patterns of α-CaMKII(T305D mutants were profoundly abnormal. Although overall firing rate, and overall burst frequency were not significantly altered in these mutants, inter-burst intervals, mean number of intra-burst spikes, ratio of intra-burst spikes to total spikes, and mean intra-burst intervals were significantly altered. In particular, the intra burst intervals of place cells in α-CaMKII(T305D mutants showed higher variability than controls. These results provide in vivo evidence that besides its well-known function in synaptic plasticity, α-CaMKII, and in particular its inhibitory phosphorylation at threonine 305, also have a role in shaping the temporal structure of hippocampal burst patterns. These results suggest that some of the molecular processes involved in acquiring information may also shape the patterns used to encode this information.

  6. Effects of Ureaplasma diversum on bovine oviductal explants: quantitative measurement using a calmodulin assay.

    Science.gov (United States)

    Smits, B; Rosendal, S; Ruhnke, H L; Plante, C; O'Brien, P J; Miller, R B

    1994-01-01

    Calmodulin (CAM) acts as an intracellular regulator of calcium, an important mediator of many cell processes. We used the CAM assay and electron microscopy to investigate the effects of Ureaplasma diversum on bovine oviductal explants obtained aseptically from slaughtered cows. A stock suspension of U. diversum (treated specimens) and sterile broth (controls) was added to replicates of cultured explants and incubated at 38 degrees C in an atmosphere of 5.5% CO2 for 48 hours. Explants were examined for ciliary activity, extracellular CAM loss, and for histological and ultrastructural changes. Explants and their culture media were examined for changes in CAM concentration. All experiments were replicated three times. In addition, U. diversum, medium and broth were assayed for CAM content. The concentrations of CAM in explants and media changed significantly (p diversum when compared to controls. The controls and infected specimens did not differ histologically or ultrastructurally, but U. diversum was seen to be closely associated with infected explant tissue. In view of this close affinity it is assumed the loss of CAM from the oviductal cells was causally related, but this was not proven. The failure to show cell membrane injury on light and electron microscopic examination was probably related to the short duration of the experiment and may only point out the sensitivity of the CAM assay in detecting early cell membrane injury. Compromise in characteristics of the medium to support both, the viability of oviductal cells and U. diversum limited the experimental time to 48 hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:8004536

  7. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Hudmon, Andy; Schulman, Howard

    2002-06-15

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a 'molecular switch-like' behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure-function of this multifunctional protein kinase. PMID:11931644

  8. Cooperativity between calmodulin-binding sites in Kv7.2 channels.

    Science.gov (United States)

    Alaimo, Alessandro; Alberdi, Araitz; Gomis-Perez, Carolina; Fernández-Orth, Juncal; Gómez-Posada, Juan Camilo; Areso, Pilar; Villarroel, Alvaro

    2013-01-01

    Among the multiple roles assigned to calmodulin (CaM), controlling the surface expression of Kv7.2 channels by binding to two discontinuous sites is a unique property of this Ca(2+) binding protein. Mutations that interfere with CaM binding or the sequestering of CaM prevent this M-channel component from exiting the endoplasmic reticulum (ER), which reduces M-current density in hippocampal neurons, enhancing excitability and offering a rational mechanism to explain some forms of benign familial neonatal convulsions (BFNC). Previously, we identified a mutation (S511D) that impedes CaM binding while allowing the channel to exit the ER, hinting that CaM binding may not be strictly required for Kv7.2 channel trafficking to the plasma membrane. Alternatively, this interaction with CaM might escape detection and, indeed, we now show that the S511D mutant contains functional CaM-binding sites that are not detected by classical biochemical techniques. Surface expression and function is rescued by CaM, suggesting that free CaM in HEK293 cells is limiting and reinforcing the hypothesis that CaM binding is required for ER exit. Within the CaM-binding domain formed by two sites (helix A and helix B), we show that CaM binds to helix B with higher apparent affinity than helix A, both in the presence and absence of Ca(2+), and that the two sites cooperate. Hence, CaM can bridge two binding domains, anchoring helix A of one subunit to helix B of another subunit, in this way influencing the function of Kv7.2 channels.

  9. Oxidized calmodulin kinase II regulates conduction following myocardial infarction: a computational analysis.

    Directory of Open Access Journals (Sweden)

    Matthew D Christensen

    2009-12-01

    Full Text Available Calmodulin kinase II (CaMKII mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ. These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.

  10. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2012-02-13

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus (= Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca 2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. © 2012 Chen et al.

  11. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    Science.gov (United States)

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  12. Human platelet calmodulin-binding proteins: Ca/sup 2 +/-dependent proteolysis upon platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1986-05-01

    Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and /sup 125/I-CaM. Ten distinct proteins with molecular weights of 245, 225K, 175K, 150K, 90K, 82K(2), 60K and 41K(2) bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner; the binding was blocked by both trifluoperazine and nonradiolabeled CaM. The 225K and 90K proteins were labeled by antisera against myosin light chain kinase (MLCK); the 60K and one of the 82K proteins were identified as the CaM-dependent phosphatase and caldesmon. The remaining proteins have not yet been identified. Most of the CaM-binding proteins were degraded upon addition of Ca/sup 2 +/ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin or N-ethyl-maleimide which suggests that it was due to a Ca/sup 2 +/-dependent protease. Activation of intact platelets by thrombin, ADP, collagen and the Ca/sup 2 +/-ionophores A23187 and ionomycin under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca/sup 2 +/) also resulted in limited proteolysis of CaM-binding proteins including those labeled with anti-MLCK and the phosphatase. Many Ca/sup 2 +//CaM-regulated enzymes have been shown to be irreversibly activated in vitro by limited proteolysis. Their data indicates that limited proteolysis also occurs in vivo; under certain conditions proteolysis may be an important physiological mechanism for irreversibly activating Ca/sup 2 +//CaM-regulated enzymes.

  13. Molecular evolution of the multiple calmodulin-like cal genes in C. elegans and in nematodes.

    Science.gov (United States)

    Karabinos, Anton

    2016-09-01

    Calmodulin (CaM) is a major EF hand containing intracellular calcium receptor in animals and plants; however, eukaryotes also express a number of related CaM-like proteins. We have previously characterized an embryonic phenotype of the single Caenorhabditis elegans CaM gene cmd-1, reported no visible RNAi phenotype for the four related cal-1 to cal-4 genes and started tissue-specific expression analyses of these proteins. In the present study, we analyzed evolutionary aspects of the previously reported CAL-1 to CAL-4 proteins, along with the four new CAL-5 to CAL-8 sequences retrieved from the worm database. Phylogenetic analyses suggest that all C. elegans CAL proteins arose from a CaM ancestor through repeated gene duplications, fusions and sequence divergence. The same holds, also, for the variable N-terminal extensions of the CAL-1 to CAL-4 proteins, which have evolved from the CaM-like core domain. We found 97 CAL homologs in different nematode clades and also detected two CAL-7-related sequences outside the nematodes. Moreover, the C. elegans-specific cal-6 gene, representing the most CaM-related sequence found in nematodes so far, harbours many deletions, insertions and sequence substitutions and is predicted, therefore, to be non-functional. These analyses provide an insight into a complex and dynamic origin of the multiple CAL genes in C. elegans and in nematodes and represent also a basis for further functional studies of these CaM-related sequences in nematodes. PMID:27558386

  14. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.

    Directory of Open Access Journals (Sweden)

    Peter Rellos

    Full Text Available UNLABELLED: Long-term potentiation (LTP, a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM-dependent kinase II (CaMKII. CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

  15. Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

    Science.gov (United States)

    Huang, J. F.; Teyton, L.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

  16. Isolation and Characterization of Calmodulin Gene of Alexandrium catenella (Dinoflagellate) and Its Performance in Cell Growth and Heat Stress

    Institute of Scientific and Technical Information of China (English)

    WEN Ruobing; SUI Zhenghong; BAO Zhenmin; ZHOU Wei; WANG Chunyan

    2014-01-01

    Harmful algal blooms (HABs) can occur and then disappear quickly, corresponding to consistent growing and declining of heavy biomasses. The molecular mechanism of blooming remains unclear. In this study, calmodulin gene (cam) of HAB causing species Alexandrium catenella was isolated and characterized. The expression of calmodulin gene was profiled at different growth rates and in heat stress. The full cDNA of cam was 597 nucleotides (nt) in length, including a 25 nt 5′untranslated region (UTR), an 122 nt 3′ UTR, and a 450 nt open reading frame (ORF) encoding 149 amino acids. The deduced calmodulin (CaM) was highly conserved in comparison with those of other organisms. As was determined with real-time RT PCR, the abundance of cam transcript varied in a pattern similar to cell growth rate during the whole growing period. The abundance of cam transcript increased by more than 8 folds from lag growth phase to exponential growth phase, and then obviously decreased from exponential growth phase to stationary/decline growth phase. In addition, the relative abundance of cam transcript significantly declined with time during heat shock. Taking CaM function described in other organisms into account, we believe that Ca2+-involved signal transduction, methyla-tion of DNA and toxin precursors underlined the cell growth of this species. The response of cam gene to heat stress in dinoflagellate suggested restrictions in Ca2+signal transduction and methylation. These findings are helpful to understand the relationships among growth, cell signal transduction, bloom formation and interaction with environmental stimuli in dinoflagellates.

  17. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone;

    2010-01-01

    of calcium-bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...... crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 A, beta = 113.2 degrees. A complete data set was collected to 3.0 A resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin....

  18. Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries

    DEFF Research Database (Denmark)

    Waldsee, Roya; Ahnstedt, Hilda; Eftekhari, Sajedeh;

    2010-01-01

    Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ET(B) receptors in smooth muscle cells via increased transcription. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CAMK......(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ET(A) and ET(B) receptors and CAMKII were determined by real-time PCR, and their protein levels were evaluated by immunohistochemistry and Western blot. The mRNA levels of CAMKII and the ET(B) receptor increased during organ...

  19. Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.

    OpenAIRE

    Tippetts, M T; Robertson, D L

    1988-01-01

    The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like th...

  20. Distinct properties of Ca2+-calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Sze-Yi; Procko, Erik; Gaudet, Rachelle [Harvard

    2012-11-01

    Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca2+–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca2+–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca2+–CaM, respectively. This structure is similar to canonical Ca2+–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca2+–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca2+–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca2+–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.

  1. Calmodulin modulates the delay period between release of calcium from internal stores and activation of calcium influx via endogenous TRP1 channels.

    Science.gov (United States)

    Vaca, Luis; Sampieri, Alicia

    2002-11-01

    In the present study we have explored the role of calmodulin (CaM) and inositol 1,4,5-trisphosphate receptor (IP(3)R) in the communication process activated after the release of calcium from the endoplasmic reticulum (ER) and the activation of calcium influx via endogenous TRP1 channels from Chinese hamster ovary cells. Experiments using combined rapid confocal calcium and electrophysiology measurements uncovered a consistent delay of around 900 ms between the first detectable calcium released from the ER and the activation of the calcium current. This delay was evident with two different methods used to release calcium from the ER: either the blockade of the microsomal calcium ATPase with thapsigargin or activation of bradykinin receptors linked to the IP(3) cascade. Direct application of IP(3) or a peptide from the NH(2)-terminal region of the IP(3)R activated store operated calcium, reducing the delay period. Introduction of CaM into the cell via the patch pipette increased the delay period from 900 +/- 100 ms to 10 +/- 2.1 s (n = 18). Furthermore, the use of selective CaM antagonists W7 and trifluoperazine maleate resulted in a substantial reduction of the delay period to 200 +/- 100 ms with 5 microm trifluoperazine maleate (n = 16) and 150 +/- 50 ms with 500 nm W7 (n = 22). CaM reduced also the current density activated by thapsigargin or brandykinin to about 60% from control. The CaM antagonists did not affect significantly the current density. The results presented here are consistent with an antagonistic effect of IP(3)R and CaM for the activation of store operated calcium after depletion of the ER. The functional competition between the activating effect of IP(3)R and the inhibiting effect of CaM may modulate the delay period between the release of calcium from the ER and the activation of calcium influx observed in different cells, as well as the amount of current activated after depletion of the ER.

  2. Antimicrobial Peptides from Plants

    Science.gov (United States)

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  3. Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    James P. Tam

    2015-11-01

    Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

  4. Electron transfer in peptides.

    Science.gov (United States)

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  5. Electromembrane extraction of peptides.

    Science.gov (United States)

    Balchen, Marte; Reubsaet, Léon; Pedersen-Bjergaard, Stig

    2008-06-20

    Rapid extraction of eight different peptides using electromembrane extraction (EME) was demonstrated for the first time. During an extraction time of 5 min, the model peptides migrated from a 500 microL aqueous acidic sample solution, through a thin supported liquid membrane (SLM) of an organic liquid sustained in the pores in the wall of a porous hollow fiber, and into a 25 microL aqueous acidic acceptor solution present inside the lumen of the hollow fiber. The driving force of the extraction was a 50 V potential sustained across the SLM, with the positive electrode in the sample and the negative electrode in the acceptor solution. The nature and the composition of the SLM were highly important for the EME process, and a mixture of 1-octanol and 15% di(2-ethylhexyl) phosphate was found to work properly. Using 1mM HCl as background electrolyte in the sample and 100 mM HCl in the acceptor solution, and agitation at 1050 rpm, enrichment up to 11 times was achieved. Recoveries were found to be dependent on the structure of the peptide, indicating that the polarity and the number of ionized groups were important parameters affecting the extraction efficiency. The experimental findings suggested that electromembrane extraction of peptides is possible and may be a valuable tool for future extraction of peptides. PMID:18479691

  6. Regulating effect of calcium ion,calmodulin and calmodulin dependent protein kinase in opioid addiction%钙离子及其结合蛋白、蛋白激酶对阿片成瘾的调节作用

    Institute of Scientific and Technical Information of China (English)

    张静

    2015-01-01

    The calcium ion is an important intracellular second messenger. The calcium ion,calmodulin and calmodulin dependent protein kinase play an important role in drug dependence,with the interaction with the mesolimbic dopamine system. Further study on calcium ion will lead to expand the understanding of the neural mechanisms underlying drug addiction,and provide an effective way for the development of low tolerance,low dependence of pain medicine as well as for the treatment of opioid addiction and relapse.%钙离子是细胞内重要的第二信使。与受体偶联的细胞内钙离子、钙调蛋白和两者依赖的钙蛋白激酶Ⅱ能够通过与中脑边缘多巴胺系统的相互作用来影响药物成瘾,在药物依赖和耐受等过程中具有重要作用。对三者的研究将指引人们对药物成瘾过程神经机制的理解,并为开发低耐受性、低依赖性镇痛药物和药物依赖的防治以及解决戒毒后的“复吸”等问题奠定基础。

  7. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders

    2015-01-01

    infection directed to subdominant conserved HIV-1 epitopes restricted to frequent HLA supertypes. The rationale for selecting HIV peptides and adjuvants are provided. Peptide subunit vaccines are regarded as safe due to the simplicity, quality, purity, and low toxicity. The caveat is reduced immunogenicity......Therapeutic vaccines aim to control chronic HIV infection and eliminate the need for lifelong antiretroviral therapy (ART). Therapeutic HIV vaccine is being pursued as part of a functional cure for HIV/AIDS. We have outlined a basic protocol for inducing new T cell immunity during chronic HIV-1...... and hence adjuvants are included to enhance and direct the immune response. Although the vaccine has been tested in ART naïve individuals, we recommend future testing of the vaccine during (early started) ART that improves immune function and to select individuals likely to benefit. Peptides representing...

  8. Synthetic antibiofilm peptides.

    Science.gov (United States)

    de la Fuente-Núñez, César; Cardoso, Marlon Henrique; de Souza Cândido, Elizabete; Franco, Octavio Luiz; Hancock, Robert E W

    2016-05-01

    Bacteria predominantly exist as multicellular aggregates known as biofilms that are associated with at least two thirds of all infections and exhibit increased adaptive resistance to conventional antibiotic therapies. Therefore, biofilms are major contributors to the global health problem of antibiotic resistance, and novel approaches to counter them are urgently needed. Small molecules of the innate immune system called host defense peptides (HDPs) have emerged as promising templates for the design of potent, broad-spectrum antibiofilm agents. Here, we review recent developments in the new field of synthetic antibiofilm peptides, including mechanistic insights, synergistic interactions with available antibiotics, and their potential as novel antimicrobials against persistent infections caused by biofilms. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. PMID:26724202

  9. Biomimetic peptide nanosensors.

    Science.gov (United States)

    Cui, Yue; Kim, Sang N; Naik, Rajesh R; McAlpine, Michael C

    2012-05-15

    The development of a miniaturized sensing platform tailored for sensitive and selective detection of a variety of biochemical analytes could offer transformative fundamental and technological opportunities. Due to their high surface-to-volume ratios, nanoscale materials are extremely sensitive sensors. Likewise, peptides represent robust substrates for selective recognition due to the potential for broad chemical diversity within their relatively compact size. Here we explore the possibilities of linking peptides to nanosensors for the selective detection of biochemical targets. Such systems raise a number of interesting fundamental challenges: What are the peptide sequences, and how can rational design be used to derive selective binders? What nanomaterials should be used, and what are some strategies for assembling hybrid nanosensors? What role does molecular modeling play in elucidating response mechanisms? What is the resulting performance of these sensors, in terms of sensitivity, selectivity, and response time? What are some potential applications? This Account will highlight our early attempts to address these research challenges. Specifically, we use natural peptide sequences or sequences identified from phage display as capture elements. The sensors are based on a variety of nanomaterials including nanowires, graphene, and carbon nanotubes. We couple peptides to the nanomaterial surfaces via traditional surface functionalization methods or self-assembly. Molecular modeling provides detailed insights into the hybrid nanostructure, as well as the sensor detection mechanisms. The peptide nanosensors can distinguish chemically camouflaged mixtures of vapors and detect chemical warfare agents with sensitivities as low as parts-per-billion levels. Finally, we anticipate future uses of this technology in biomedicine: for example, devices based on these sensors could detect disease from the molecular components in human breath. Overall, these results provide a

  10. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  11. Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

    Directory of Open Access Journals (Sweden)

    J.B.R. Rodriguez

    2013-03-01

    Full Text Available Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endoplasmic reticulum Ca2+-ATPase isoform (SERCA is expressed in rat vas deferens (RVD and its modulation by calmodulin (CaM-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM and a low sensitivity to vanadate (IC50 = 41 µM. These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

  12. Calmodulin Kinase II Interacts with the Dopamine Transporter C Terminus to Regulate Amphetamine-Induced Reverse Transport

    DEFF Research Database (Denmark)

    Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion;

    2006-01-01

    Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d......Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound...... to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation...... of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux...

  13. Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport

    DEFF Research Database (Denmark)

    Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion;

    2006-01-01

    Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d......Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound...... to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation...... of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux...

  14. Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

    Science.gov (United States)

    Lu, Y. T.; Hidaka, H.; Feldman, L. J.

    1996-01-01

    Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

  15. In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

    Science.gov (United States)

    Veluthambi, K.; Poovaiah, B. W.

    1986-01-01

    In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

  16. Dicyclopropylmethyl peptide backbone protectant.

    Science.gov (United States)

    Carpino, Louis A; Nasr, Khaled; Abdel-Maksoud, Adel Ali; El-Faham, Ayman; Ionescu, Dumitru; Henklein, Peter; Wenschuh, Holger; Beyermann, Michael; Krause, Eberhard; Bienert, Michael

    2009-08-20

    The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

  17. Invertebrate FMRFamide related peptides.

    Science.gov (United States)

    Krajniak, Kevin G

    2013-06-01

    In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

  18. Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein.

    Science.gov (United States)

    O'Day, Danton H; Chatterjee-Chakraborty, Munmun; Wagler, Stephanie; Myre, Michael A

    2005-06-17

    Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein. PMID:15883042

  19. Driving Calmodulin Protein towards Conformational Shift by Changing Ionization States of Select Residues

    Science.gov (United States)

    Negi, Sunita; Rana Atilgan, Ali; Atilgan, Canan

    2012-12-01

    Proteins are complex systems made up of many conformational sub-states which are mainly determined by the folded structure. External factors such as solvent type, temperature, pH and ionic strength play a very important role in the conformations sampled by proteins. Here we study the conformational multiplicity of calmodulin (CaM) which is a protein that plays an important role in calcium signaling pathways in the eukaryotic cells. CaM can bind to a variety of other proteins or small organic compounds, and mediates different physiological processes by activating various enzymes. Binding of calcium ions and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner. In particular, we discuss the effect of pH variation on the conformations of CaM. By using the pKa values of the charged residues as a basis to assign protonation states, the conformational changes induced in CaM by reducing the pH are studied by molecular dynamics simulations. Our current view suggests that at high pH, barrier crossing to the compact form is prevented by repulsive electrostatic interactions between the two lobes. At reduced pH, not only is barrier crossing facilitated by protonation of residues, but also conformations which are on average more compact are attained. The latter are in accordance with the fluorescence resonance energy transfer experiment results of other workers. The key events leading to the conformational change from the open to the compact conformation are (i) formation of a salt bridge between the N-lobe and the linker, stabilizing their relative motions, (ii) bending of the C-lobe towards the N-lobe, leading to a lowering of the interaction energy between the two-lobes, (iii) formation of a hydrophobic patch between the two lobes, further stabilizing the bent conformation by reducing the entropic cost of the compact form, (iv) sharing of a Ca+2 ion between the two lobes.

  20. Particulate air pollution induces arrhythmia via oxidative stress and calcium calmodulin kinase II activation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin-Bae [The Division of Cardiology, Kyung Hee University College of Medicine, 1 Hoegi-dong, Dongdaemun-Gu, Seoul (Korea, Republic of); Kim, Changsoo [The Department of Preventive Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Choi, Eunmi [Cardiovascular Research Institute and Severance Biomedical Science Institute, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Park, Sanghoon; Park, Hyelim; Pak, Hui-Nam; Lee, Moon-Hyoung [The Division of Cardiology, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Shin, Dong Chun [The Department of Preventive Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Hwang, Ki-Chul [Cardiovascular Research Institute and Severance Biomedical Science Institute, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); The Division of Cardiology, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Joung, Boyoung, E-mail: cby6908@yuhs.ac [The Division of Cardiology, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of)

    2012-02-15

    Ambient particulate matter (PM) can increase the incidence of arrhythmia. However, the arrhythmogenic mechanism of PM is poorly understood. This study investigated the arrhythmogenic mechanism of PM. In Sprague–Dawley rats, QT interval was increased from 115.0 ± 14.0 to 142.1 ± 18.4 ms (p = 0.02) after endotracheal exposure of DEP (200 μg/ml for 30 min, n = 5). Ventricular premature contractions were more frequently observed after DEP exposure (100%) than baseline (20%, p = 0.04). These effects were prevented by pretreatment of N-acetylcysteine (NAC, 5 mmol/L, n = 3). In 12 Langendorff-perfused rat hearts, DEP infusion of 12.5 μg/ml for 20 min prolonged action potential duration (APD) at only left ventricular base increasing apicobasal repolarization gradients. Spontaneous early afterdepolarization (EAD) and ventricular tachycardia (VT) were observed in 8 (67%) and 6 (50%) hearts, respectively, versus no spontaneous triggered activity or VT in any hearts before DEP infusion. DEP-induced APD prolongation, EAD and VT were successfully prevented with NAC (5 mmol/L, n = 5), nifedipine (10 μmol/L, n = 5), and active Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) blockade, KN 93 (1 μmol/L, n = 5), but not by thapsigargin (200 nmol/L) plus ryanodine (10 μmol/L, n = 5) and inactive CaMKII blockade, KN 92 (1 μmol/L, n = 5). In neonatal rat cardiomyocytes, DEP provoked ROS generation in dose dependant manner. DEP (12.5 μg/ml) induced apoptosis, and this effect was prevented by NAC and KN 93. Thus, this study shows that in vivo and vitro exposure of PM induced APD prolongation, EAD and ventricular arrhythmia. These effects might be caused by oxidative stress and CaMKII activation. -- Highlights: ► The ambient PM consistently prolonged repolarization. ► The ambient PM induced triggered activity and ventricular arrhythmia. ► These effects were prevented by antioxidants, I{sub CaL} blockade and CaMKII blockade. ► The ambient PM can induce

  1. Volatile anesthetics inhibit the activity of calmodulin by interacting with its hydrophobic site

    Institute of Scientific and Technical Information of China (English)

    ZHOU Miao-miao; XIA Hui-min; LIU Jiao; XU You-nian; XIN Nai-xin; ZHANG Shi-hai

    2012-01-01

    Background Volatile anesthetics (VAs) may affect varied and complex physiology processes by manipulating Ca2+-calmodulin (CaM).However,the detailed mechanism about the action of VAs on CaM has not been elucidated.This study was undertaken to examine the effects of VAs on the conformational change,hydrophobic site,and downstream signaling pathway of CaM,to explore the possible mechanism of anesthetic action of VAs.Methods Real-time second-harmonic generation (SHG) was performed to monitor the conformational change of CaM in the presence of VAs, each plus 100 μmol/L Ca2+. A hydrophobic fluorescence indicator,8-anilinonaphthalene-1-sulfonate (ANS),was utilized to define whether the VAs would interact with CaM at the hydrophobic site or not.High-performance liquid chromatography (HPLC) was carried out to analyze the activity of CaM-dependent phosphodiesterase (PDE1) in the presence of VAs.The VAs studied were ether,enflurane,isoflurane,and sevoflurane,with their aqueous concentrations 7.6,9.5,11.4 mmol/L; 0.42,0.52,0.62 mmol/L; 0.25,0.31,0.37 mmol/L and 0.47,0.59,0.71 mmol/L respectively,each were equivalent to their 0.8,1.0 and 1.2 concentration for 50% of maximal effect (EC50) for general anesthesia.Results The second-harmonic radiation of CaM in the presence of Ca2+ was largely inhibited by the VAs.The fluorescence intensity of ANS,generated by binding of Ca2+ to CaM,was reversed by the VAs.HPLC results also showed that AMP,the product of the hydrolysis of cAMP by CaM-dependent PDE1,was reduced by the VAs.Conclusions Our findings demonstrate that the above VAs interact with the hydrophobic core of Ca2+-CaM and the interaction results in the inhibition of the conformational change and activity of CaM.This in vitro study may provide us insight into the possible mechanism of anesthetic action of VAs in vivo.

  2. Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

    Directory of Open Access Journals (Sweden)

    Sitvanit Haziza

    2015-08-01

    Full Text Available Calmodulin lysine methyl transferase (CaM KMT is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence

  3. Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

    Science.gov (United States)

    Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L; Sheffield, Val C; Golan, Hava; Parvari, Ruti

    2015-08-01

    Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the Ca

  4. Natriuretic peptides and cerebral hemodynamics

    DEFF Research Database (Denmark)

    Guo, Song; Barringer, Filippa; Zois, Nora Elisabeth;

    2014-01-01

    Natriuretic peptides have emerged as important diagnostic and prognostic tools for cardiovascular disease. Plasma measurement of the bioactive peptides as well as precursor-derived fragments is a sensitive tool in assessing heart failure. In heart failure, the peptides are used as treatment...

  5. Descriptors for antimicrobial peptides

    DEFF Research Database (Denmark)

    Jenssen, Håvard

    2011-01-01

    Introduction: A frightening increase in the number of isolated multidrug resistant bacterial strains linked to the decline in novel antimicrobial drugs entering the market is a great cause for concern. Cationic antimicrobial peptides (AMPs) have lately been introduced as a potential new class of ...

  6. Isolation, characterization, and bioinformatic analysis of calmodulin-binding protein cmbB reveals a novel tandem IP22 repeat common to many Dictyostelium and Mimivirus proteins.

    Science.gov (United States)

    O'Day, Danton H; Suhre, Karsten; Myre, Michael A; Chatterjee-Chakraborty, Munmun; Chavez, Sara E

    2006-08-01

    A novel calmodulin-binding protein cmbB from Dictyostelium discoideum is encoded in a single gene. Northern analysis reveals two cmbB transcripts first detectable at 4 h during multicellular development. Western blotting detects an approximately 46.6 kDa protein. Sequence analysis and calmodulin-agarose binding studies identified a "classic" calcium-dependent calmodulin-binding domain (179IPKSLRSLFLGKGYNQPLEF198) but structural analyses suggest binding may not involve classic alpha-helical calmodulin-binding. The cmbB protein is comprised of tandem repeats of a newly identified IP22 motif ([I,L]Pxxhxxhxhxxxhxxxhxxxx; where h = any hydrophobic amino acid) that is highly conserved and a more precise representation of the FNIP repeat. At least eight Acanthamoeba polyphaga Mimivirus proteins and over 100 Dictyostelium proteins contain tandem arrays of the IP22 motif and its variants. cmbB also shares structural homology to YopM, from the plague bacterium Yersenia pestis. PMID:16777069

  7. Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

    Science.gov (United States)

    Maubach, Gunter; Sokolova, Olga; Wolfien, Markus; Rothkötter, Hermann-Josef; Naumann, Michael

    2013-09-15

    Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

  8. Molecular characterization of a calmodulin gene, VcCaM1, that is differentially expressed under aluminum stress in highbush blueberry

    Science.gov (United States)

    Calmodulin (CaM), a small acidic protein, is one of the best characterized Ca2+ sensors in eukaryotes. This Ca2+-regulated protein plays a critical role in decoding and transducing environmental stress signals by activating specific targets. Many environmental stresses elicit changes in intracellu...

  9. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  10. Biochemical functionalization of peptide nanotubes with phage displayed peptides.

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering. PMID:27479451

  11. Radiolabelled peptides for oncological diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Laverman, Peter; Boerman, Otto C.; Oyen, Wim J.G. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Sosabowski, Jane K. [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom)

    2012-02-15

    Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The {sup 111}In-labelled somatostatin analogue octreotide (OctreoScan trademark) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours. (orig.)

  12. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  13. Antimicrobial peptides in Echinoderms

    Directory of Open Access Journals (Sweden)

    C Li

    2010-05-01

    Full Text Available Antimicrobial peptides (AMPs are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. Here we summarize the knowledge of such peptides in echinoderms. Strongylocins are a novel family of cysteine-rich AMPs, recently identified in the sea urchins, Strongylocentrotus droebachiensis and S. purpuratus. Although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, dissimilar to other known AMPs. A family of heterodimeric AMPs, named centrocins, are also present in S. droebachiensis. Lysozymes and fragments of larger proteins, such as beta-thymocins, actin, histone 2A and filamin A have also been shown to display antimicrobial activities in echinoderms. Future studies on AMPs should be aimed in revealing how echinoderms use these AMPs in the immune response against microbial pathogens.

  14. Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations

    DEFF Research Database (Denmark)

    Shamgar, Liora; Ma, Lijuan; Schmitt, Nicole;

    2006-01-01

    The slow IKS K+ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations...... located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca(2+)-CaM that prevents...... the risk of ventricular arrhythmias. Udgivelsesdato: 2006-Apr-28...

  15. Identification of a Denitrase Activity Against Calmodulin in Activated Macrophages Using High-Field Liquid Chromatography - FTICR Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Lourette, Natacha M.; Boschek, Curt B.; Bigelow, Diana J.; Smith, Richard D.; Pasa-Tolic, Liljiana; Squier, Thomas C.

    2007-09-18

    We have identified a denitrase activity in macrophages that is upregulated following macrophage activation, which is shown by mass spectrometry to recognize nitrotyrosines in the calcium signaling protein calmodulin (CaM) and convert them to their native tyrosine structure without the formation of any aminotyrosine. Comparable extents of methionine sulfoxide reduction are also observed that are catalyzed by endogenous methionine sulfoxide reductases. Competing with repair processes, oxidized CaM is a substrate for a peptidase activity that results in the selective cleavage of the C-terminus lysine (i.e., Lys148) that is expected to diminish CaM function. Thus, competing repair and peptidase activities define the abundances and functionality of CaM to modulate cellular metabolism in response to oxidative stress, where the presence of the truncated CaM species provides a useful biomarker for the transient appearance of oxidized CaM.

  16. Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin2.

    Science.gov (United States)

    Karlsson, Göran; Persson, Cecilia; Mayzel, Maxim; Hedenström, Mattias; Backman, Lars

    2016-04-01

    Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker. PMID:26800385

  17. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    Science.gov (United States)

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

  18. Investigation of Neuronal Cell Type-Specific Gene Expression of Ca2+/Calmodulin-dependent Protein Kinase II.

    Directory of Open Access Journals (Sweden)

    Mima Kazuko

    2002-01-01

    Full Text Available The promoter activity of the rat Ca2+/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5'-flanking region of &agr; and &bgr; isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5'-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types.

  19. Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker.

    Science.gov (United States)

    Bradley, Luke H; Bricken, Michael L; Randle, Charlotte

    2011-02-01

    Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: all library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

  20. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    Science.gov (United States)

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  1. Cross-talk between calcium-calmodulin and nitric oxide in abscisic acid signaling in leaves of maize plants

    Institute of Scientific and Technical Information of China (English)

    Jianrong Sang; Aying Zhang; Fan Lin; Mingpu Tan; Mingyi Jiang

    2008-01-01

    Using pharmacological and biochemical approaches,the signaling pathways between hydrogen peroxide (H2O2),calcium (Ca2+)-calmodulin (CAM),and nitric oxide (NO) in abscisic acid (ABA)-induced antioxidant defense were investigated in leaves of maize (Zea mays L.) plants.Treatments with ABA,H2O2,and CaCI2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase (NOS) in the cytosolic and microsomal fractions of maize leaves.However,such increases were blocked by the pretreatments with Ca2+ inhibitors and CaM antagonists.Meanwhile,pretreatments with two NOS inhibitors also suppressed the Ca2+-induced increase in the production of NO.On the other hand,treatments with ABA and the NO donor sodium nitroprusside (SNP) also led to increases in the concentration of cytosolic Ca2+ in protoplasts of mesophyll cells and in the expression of calmodulin 1 (CaMI) gene and the contents of CaM in leaves of maize plants,and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor.Moreover,SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 (SOD4),cytosolic ascorbate peroxidase (cAPX),and glutathione reductase 1 (GRI) and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca2+ inhibitors and CaM antagonists.Our results suggest that Ca2+-CaM functions both upstream and downstream of NO production,which is mainly from NOS,in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.

  2. Rat vas deferens SERCA2 is modulated by Ca{sup 2+}/calmodulin protein kinase II-mediated phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.B.R.; Muzi-Filho, H. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Valverde, R.H.F. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Quintas, L.E.M. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Noel, F. [Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Einicker-Lamas, M. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ (Brazil); Cunha, V.M.N. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2013-03-19

    Ca{sup 2+} pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca{sup 2+}-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca{sup 2+} (Ca{sub 0.5} = 780 nM) and a low sensitivity to vanadate (IC{sub 50} = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca{sup 2+} and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca{sup 2+} accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca{sup 2+} and CaM, possibly via CaMKII, in a process that results in stimulation of Ca{sup 2+} pumping activity.

  3. The PeptideAtlas Project

    OpenAIRE

    Deutsch, Eric W.

    2010-01-01

    PeptideAtlas is a multi-species compendium of peptides observed with tandem mass spectrometry methods. Raw mass spectrometer output files are collected from the community and reprocessed through a uniform analysis and validation pipeline that continues to advance. The results are loaded into a database and the information derived from the raw data is returned to the community via several web-based data exploration tools. The PeptideAtlas resource is useful for experiment planning, improving g...

  4. Human Antimicrobial Peptides and Proteins

    OpenAIRE

    Guangshun Wang

    2014-01-01

    As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified ...

  5. Peptides that influence membrane topology

    Science.gov (United States)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  6. NCAM Mimetic Peptides: An Update

    DEFF Research Database (Denmark)

    Berezin, Vladimir; Bock, Elisabeth

    2008-01-01

    sequences contain one or several NCAM homophilic binding sites involved in NCAM binding to itself, have been identified. By means of NMR titration analysis and molecular modeling a number of peptides derived from NCAM and targeting NCAM heterophilic ligands such as the fibroblast growth factor receptor...... and heparan sulfate proteoglycans (HSPG) have been identified. The FGL, dekaCAM, FRM/EncaminA, BCL, EncaminC and EncaminE peptides all target the FGF receptor whereas the heparin binding peptide HBP targets HSPG. Moreover, a number of NCAM binding peptides have been identified employing screening...

  7. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology. PMID:26279082

  8. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  9. Biodiscovery of aluminum binding peptides

    Science.gov (United States)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  10. Polyclonal Peptide Antisera.

    Science.gov (United States)

    Pihl, Tina H; Illigen, Kristin E; Houen, Gunnar

    2015-01-01

    Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages. The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare. Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented. PMID:26424267

  11. 钙调素-人胰岛素原融合体的酶切加工%ENZYMATIC DIGESTION OF CALMODULIN-PROINSULIN FUSION PROTEIN

    Institute of Scientific and Technical Information of China (English)

    王学祥; 井健

    2011-01-01

    A fusion protein composed of human proinsuiin mutant and calmodulin was prepared, then digested with PRESCI protease. Digested product includes calmodulin and human proinsulin mutant. Human proinsulin mutant was purified.%通过基因工程方法将人胰岛素原突变体偶联钙调索形成融合态重组蛋白质,在该重组蛋白质中设计具有PRESCI蛋白酶酶切位点.制备PRESCI蛋白酶并对钙调素-人胰岛素原突变体重组蛋白质进行酶切加工.研究表明,该重组融合蛋白质可被有效切割,通过蛋白质纯化手段能够有效地将切割后的钙调素与人胰岛素原突变体进行纯化.

  12. Phosphorylation and activation of nuclear Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca{sup 2+}/calmodulin-dependent protein kinase I (CaMKI)

    Energy Technology Data Exchange (ETDEWEB)

    Onouchi, Takashi [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Ishida, Atsuhiko [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521 (Japan); Kameshita, Isamu [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer CaMKP-N/PPM1E underwent proteolytic processing and translocated to cytosol. Black-Right-Pointing-Pointer The proteolysis was effectively inhibited by the proteasome inhibitors. Black-Right-Pointing-Pointer Ser-480 of zebrafish CaMKP-N was phosphorylated by cytosolic CaMKI. Black-Right-Pointing-Pointer Phosphorylation-mimic mutants of CaMKP-N showed enhanced activity. Black-Right-Pointing-Pointer These results suggest that CaMKP-N is regulated by CaMKI. -- Abstract: Nuclear Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca{sup 2+}/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.

  13. Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

    OpenAIRE

    Lakowicz, J.R.; Gryczynski, I.; Laczko, G; Wiczk, W; Johnson, M.L.

    1994-01-01

    We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distance...

  14. Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase

    Science.gov (United States)

    Vitart, V.; Christodoulou, J.; Huang, J. F.; Chazin, W. J.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

  15. Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

    Science.gov (United States)

    Zhang, Yi-Le; Han, Zhao-Feng; Sun, Ying-Pu

    2016-06-01

    The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d = 25.2 ± 4.6 and 0.47 ± 0.08 µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total = -7.6 and -9.2 kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to

  16. Radiolabelled peptides for oncological diagnosis.

    NARCIS (Netherlands)

    Laverman, P.; Sosabowski, J.K.; Boerman, O.C.; Oyen, W.J.G.

    2012-01-01

    Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of resea

  17. Urinary Peptides in Rett Syndrome.

    Science.gov (United States)

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  18. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptide...

  19. Solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Jensen, Knud Jørgen

    2013-01-01

    This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective....

  20. Targeting the Small- and Intermediate-Conductance Ca2+-Activated Potassium Channels: The Drug-Binding Pocket at the Channel/Calmodulin Interface

    Directory of Open Access Journals (Sweden)

    Meng Cui

    2014-10-01

    Full Text Available The small- and intermediate-conductance Ca2+-activated potassium (SK/IK channels play important roles in the regulation of excitable cells in both the central nervous and cardiovascular systems. Evidence from animal models has implicated SK/IK channels in neurological conditions such as ataxia and alcohol use disorders. Further, genome-wide association studies have suggested that cardiovascular abnormalities such as arrhythmias and hypertension are associated with single nucleotide polymorphisms that occur within the genes encoding the SK/IK channels. The Ca2+ sensitivity of the SK/IK channels stems from a constitutively bound Ca2+-binding protein: calmodulin. Small-molecule positive modulators of SK/IK channels have been developed over the past decade, and recent structural studies have revealed that the binding pocket of these positive modulators is located at the interface between the channel and calmodulin. SK/IK channel positive modulators can potentiate channel activity by enhancing the coupling between Ca2+ sensing via calmodulin and mechanical opening of the channel. Here, we review binding pocket studies that have provided structural insight into the mechanism of action for SK/IK channel positive modulators. These studies lay the foundation for structure-based drug discovery efforts that can identify novel SK/IK channel positive modulators. © 2014 S. Karger AG, Basel

  1. Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements.

    Science.gov (United States)

    Kukic, Predrag; Lundström, Patrik; Camilloni, Carlo; Evenäs, Johan; Akke, Mikael; Vendruscolo, Michele

    2016-01-12

    Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca(2+)-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, (15)N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca(2+) by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca(2+)-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

  2. Conus venom peptide pharmacology.

    Science.gov (United States)

    Lewis, Richard J; Dutertre, Sébastien; Vetter, Irina; Christie, MacDonald J

    2012-04-01

    Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies. PMID:22407615

  3. Potential of phage-displayed peptide library technology to identify functional targeting peptides

    Science.gov (United States)

    Krumpe, Lauren RH; Mori, Toshiyuki

    2010-01-01

    Combinatorial peptide library technology is a valuable resource for drug discovery and development. Several peptide drugs developed through phage-displayed peptide library technology are presently in clinical trials and the authors envision that phage-displayed peptide library technology will assist in the discovery and development of many more. This review attempts to compile and summarize recent literature on targeting peptides developed through peptide library technology, with special emphasis on novel peptides with targeting capacity evaluated in vivo. PMID:20150977

  4. Metal binding discrimination of the calmodulin Q41C/K75C mutant on Ca2+ and La3+

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Calmodulin (CaM) is a multifunctional Ca2+-binding protein regulating the activity of many enzymes in response to fluctuation of the intracellular Ca2+ level. It has been shown that a CaM Q41C/K75C mutant (CaMSS) with a disulfide bond in the N-terminal domain exhibits greatly reduced affinity to Ca2+. In the present study, the experimental results revealed a unique metal binding pattern in CaMSS towards La3+ and Ca2+ separately: the mutant protein binds Ca2+ at site Ⅰ, Ⅲ and IV; however, it binds La3+ at site Ⅰ, Ⅱ and IV. A putative mechanism was proposed which is the conformation of site Ⅱ (or siteⅢ) of CaMSS could be altered and thus loses its metal ion affinity in response to metal binding in the opposite terminal domain possibly through the long range domain interaction. The present work may offer new perspectives for understanding the mechanisms of specific metal ion affinity in CaM and for CaM-based protein design.

  5. Graded Ca2+/calmodulin-dependent coupling of voltage-gated CaV1.2 channels

    Science.gov (United States)

    Dixon, Rose E; Moreno, Claudia M; Yuan, Can; Opitz-Araya, Ximena; Binder, Marc D; Navedo, Manuel F; Santana, Luis F

    2015-01-01

    In the heart, reliable activation of Ca2+ release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca2+ to calmodulin (CaM) and proceed through Ca2+/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. Our findings suggest a model for CaV1.2 channel gating and Ca2+-influx amplification that unifies diverse observations about Ca2+ signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently. DOI: http://dx.doi.org/10.7554/eLife.05608.001 PMID:25714924

  6. 2D FT-ICR MS of Calmodulin: A Top-Down and Bottom-Up Approach

    Science.gov (United States)

    Floris, Federico; van Agthoven, Maria; Chiron, Lionel; Soulby, Andrew J.; Wootton, Christopher A.; Lam, Yuko P. Y.; Barrow, Mark P.; Delsuc, Marc-André; O'Connor, Peter B.

    2016-09-01

    Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments.

  7. Structural Properties of Human CaMKII Ca2+ /Calmodulin-Dependent Protein Kinase II using X-ray Crystallography

    Science.gov (United States)

    Cao, Yumeng Melody; McSpadden, Ethan; Kuriyan, John; Department of Molecular; Cell Biology; Department of Chemistry Team

    To this day, human memory storage remains a mystery as we can at most describe the process vaguely on a cellular level. Switch-like properties of Calcium/Calmodulin-Dependent Protein Kinase II make it a leading candidate in understanding the molecular basis of human memory. The protein crystal was placed in the beam of a synchrotron source and the x-ray crystallography data was collected as reflections on a diffraction pattern that undergo Fourier transform to obtain the electron density. We observed two drastic differences from our solved structure at 2.75Å to a similar construct of the mouse CaMKII association domain. Firstly, our structure is a 6-fold symmetric dodecamer, whereas the previously published construct was a 7-fold symmetric tetradecamer. This suggests the association domain of human CaMKII is a dynamic structure that is triggered subunit exchange process. Secondly, in our structure the N-terminal tag is docked as an additional beta-strand on an uncapped beta-sheet present in each association domain protomer. This is concrete evidence of the involvement of the polypeptide docking site in the molecular mechanism underlining subunit exchange. In the future, we would like to selectively inhibit the exchange process while not disrupting the other functionalities of CaMKII.

  8. Intrathecal inhibition of calcium/calmodulin-dependent protein kinase II in diabetic neuropathy adversely affects pain-related behavior.

    Science.gov (United States)

    Jelicic Kadic, Antonia; Boric, Matija; Ferhatovic, Lejla; Banozic, Adriana; Sapunar, Damir; Puljak, Livia

    2013-10-25

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy. PMID:24035897

  9. The Role of Extracellular Ca2+ Influx,Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

    Institute of Scientific and Technical Information of China (English)

    SunQing-yuan; TanJing-he; 等

    1999-01-01

    The effects of various Ca2+-modifying drugs on moue egg fertilization were studied.Ca2+ chelator,ethylen glycol-bis-(2-aminoethyl)-tetracetic acid(EGTA),and calmodulin(CaM) antagonist,trifluoperzaine (TFP),inhibited fertilization in a dose-dependent manner,whild Ca2+ channel bolcker,verspamil,did not have any effect.When intracellular Ca2+ release was blocked by 8-(N,N-diethylamino) octy 1-3,4,5-trimethoxy-benzonate(TME-8) or the Ca2+ oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca2+-At-Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased.In contrast,inhibition of intracellular Ca2+ release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization.The results sugest that both extracellular influx,intracellular Ca2+ release and CaM activation are required for mormal fertilization.However,extracellular influx through voltage-gated Ca2+ channel and intracellular release induced by IP3 and not the only pathways for producing Ca2+ transients in moue eggs.

  10. The Role of Extracellular Ca2+Influx, Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    The effects of various Ca2+-modifying drugs on moue egg fertilization were studied. Ca2+ chelator, ethylen glycol-bis-(2-aminoethyl)-tetracetic acid (EGTA) ,and calmodulin (CaM) antagonist,trifluoperzaine (TFP) ,inhibited fertilization in a dose-dependent manner,whild Ca2+ channel bolcker,verapamil ,did not have any effect. When intracellular Ca2+ release was blocked by 8-(N, N-diethylamino) octy1-3,4,5-trimethoxy- benzonate (TMB-8) or the Ca2+ oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca2+-AT- Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased. In contrast,inhibition of intracellular Ca2+ release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization. The results sugest that both extracellular influx,intracellu- lar Ca2+ release and CaM activation are required for normal fertilization. However ,extracellular influx through voltage-gated Ca2+ channel and intracellular release induced by IP3 are not the only pathways for producing Ca2+ transients in moue eggs.

  11. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

    Science.gov (United States)

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H

    1996-06-17

    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

  12. Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution.

    Science.gov (United States)

    Zik, M; Arazi, T; Snedden, W A; Fromm, H

    1998-08-01

    The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

  13. The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.

    Science.gov (United States)

    Rodríguez-Concepción, M; Yalovsky, S; Zik, M; Fromm, H; Gruissem, W

    1999-04-01

    Post-translational attachment of isoprenyl groups to conserved cysteine residues at the C-terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C-terminal basic domain and a CTIL CaaX-box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild-type CaM53 or a non-prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non-prenylated mutant protein localizes to the nucleus, indicating a dual role for the C-terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities.

  14. Pavlovian fear conditioning regulates Thr286 autophosphorylation of Ca2+/calmodulin-dependent protein kinase II at lateral amygdala synapses.

    Science.gov (United States)

    Rodrigues, Sarina M; Farb, Claudia R; Bauer, Elizabeth P; LeDoux, Joseph E; Schafe, Glenn E

    2004-03-31

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in synaptic plasticity and memory formation in a variety of learning systems and species. The present experiments examined the role of CaMKII in the circuitry underlying pavlovian fear conditioning. First, we reveal by immunocytochemical and tract-tracing methods that alphaCaMKII is postsynaptic to auditory thalamic inputs and colocalized with the NR2B subunit of the NMDA receptor. Furthermore, we show that fear conditioning results in an increase of the autophosphorylated (active) form of alphaCaMKII in lateral amygdala (LA) spines. Next, we demonstrate that intra-amygdala infusion of a CaMK inhibitor, 1-[NO-bis-1,5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl-4-phenylpiperazine, KN-62, dose-dependently impairs the acquisition, but not the expression, of auditory and contextual fear conditioning. Finally, in electrophysiological experiments, we demonstrate that an NMDA receptor-dependent form of long-term potentiation at thalamic input synapses to the LA is impaired by bath application of KN-62 in vitro. Together, the results of these experiments provide the first comprehensive view of the role of CaMKII in the amygdala during fear conditioning.

  15. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    2015-07-01

    Full Text Available Bacillus anthracis adenylyl cyclase toxin edema factor (EF is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH-oxidase, thus reducing production of reactive oxygen species (ROS used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut with Met to leucine (Leu substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

  16. Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

    Science.gov (United States)

    Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

    1995-01-01

    Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

  17. Radiopharmaceutical development of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

    Fani, Melpomeni; Maecke, Helmut R. [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany)

    2012-02-15

    Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as {sup 99m}Tc, M{sup 3+} radiometals ({sup 111}In, {sup 86/90}Y, {sup 177}Lu, {sup 67/68}Ga), {sup 64/67}Cu, {sup 18}F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin

  18. Peptide primary messengers in plants

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The peptide primary messengers regulate embryonic development,cell growth and many other activities in animal cells. But recent evidence verified that peptide primary messengers are also involved in plant defense responses, the recognition between pollen and stigma and keep the balance between cell proliferation and differentiations in shoot apical meristems. Those results suggest that plants may actually make wide use of peptide primary messengers, both in embryonic development and late life when they rally their cells to defend against pathogens and insect pests. The recent advance in those aspects is reviewed.

  19. Screening of TACE Peptide Inhibitors from Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.

  20. Effects of Rare Earth Ions on the Interaction between Calmodulin and Melittin%稀土离子对钙调蛋白与蜂毒素作用的影响

    Institute of Scientific and Technical Information of China (English)

    李伟国; 张杰; 赵大庆; 倪嘉缵

    2001-01-01

    The effects of terbium ion on the conformation of calmodulin and on the interaction between calmodulin and melittin have been studied by the endogenous fluorescent spectrometry of calmodulin and melittin,and the sensitized fluorescent spectrometry of terbium ion,respectively.The results show that terbium ions have a tight binding site in the I and II metal-binding sites of calmodulin.The conformation of calmodulin induced by terbium ion can bind melittin and transfer the tryptophane residue of melittin to a relatively hydrophobic environment,while the binding of melittin to calmodulin produces effect on the binding orders of terbium ion in camodulin.Results from FT-IR spectrometry have revealed that upon binding of lanthanum ion,apo-calmodulin undergoes a conformational change with the increase of α-helix content and the decrease of β-sheet content.Melittin's binding to calmodulin has no effect on its conformation induced by the binding of lanthanum ion to calmodulin.%分别用钙调蛋白和蜂毒素的内源荧光光谱以及铽离子的敏化荧光光谱考察了铽离子对钙调蛋白构象变化以及对钙调蛋白与蜂毒素相互作用的影响.结果表明,铽离子首先结合在钙调蛋白的第Ⅰ和第Ⅱ位点,铽离子不影响钙调蛋白与蜂毒素的相互作用,蜂毒素与钙调蛋白作用后不影响铽离子在钙调蛋白上的键合顺序.傅里叶变换红外光谱结果表明三价的镧离子与钙调蛋白作用使钙调蛋白的α螺旋结构增加,β折叠结构减少,与钙离子对它的二级结构影响相类似.稀土离子在钙调蛋白-蜂毒素复合体系中主要与钙调蛋白作用.

  1. New vasoactive peptides in cirrhosis

    DEFF Research Database (Denmark)

    Kimer, Nina; Goetze, Jens Peter; Bendtsen, Flemming;

    2014-01-01

    BACKGROUND: Patients with cirrhosis have substantial circulatory imbalance between vasoconstrictive and vasodilating forces. The study of circulatory vasoactive peptides may provide important pathophysiological information. This study aimed to assess concentrations, organ extraction and relations...

  2. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte;

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...... improve biomolecular recognition by synthetic nucleic acid analogues. Circular dichroism (CD) measurements showed no distortion of the duplex structure by the incorporated peptide chains while studies in human serum indicated superior stability of the POCs compared to LNA/DNA mixmers and unmodified DNA...

  3. Peptide nanostructures in biomedical technology.

    Science.gov (United States)

    Feyzizarnagh, Hamid; Yoon, Do-Young; Goltz, Mark; Kim, Dong-Shik

    2016-09-01

    Nanostructures of peptides have been investigated for biomedical applications due to their unique mechanical and electrical properties in addition to their excellent biocompatibility. Peptides may form fibrils, spheres and tubes in nanoscale depending on the formation conditions. These peptide nanostructures can be used in electrical, medical, dental, and environmental applications. Applications of these nanostructures include, but are not limited to, electronic devices, biosensing, medical imaging and diagnosis, drug delivery, tissue engineering and stem cell research. This review offers a discussion of basic synthesis methods, properties and application of these nanomaterials. The review concludes with recommendations and future directions for peptide nanostructures. WIREs Nanomed Nanobiotechnol 2016, 8:730-743. doi: 10.1002/wnan.1393 For further resources related to this article, please visit the WIREs website. PMID:26846352

  4. Targeting cancer with peptide aptamers

    OpenAIRE

    Seigneuric, Renaud; Gobbo, Jessica; Colas, Pierre; Garrido, Carmen

    2011-01-01

    A major endeavour in cancer chemotherapy is to develop agents that specifically target a biomolecule of interest. There are two main classes of targeting agents: small molecules and biologics. Among biologics (e.g.: antibodies), DNA, RNA but also peptide aptamers are relatively recent agents. Peptide aptamers are seldom described but represent attractive agents that can inhibit a growing panel of oncotargets including Heat Shock Proteins. Potential pitfalls and coming challenges towards succe...

  5. Peptides and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  6. Kinins and peptide receptors.

    Science.gov (United States)

    Regoli, Domenico; Gobeil, Fernand

    2016-04-01

    This paper is divided into two sections: the first contains the essential elements of the opening lecture presented by Pr. Regoli to the 2015 International Kinin Symposium in S. Paulo, Brazil on June 28th and the second is the celebration of Dr. Regoli's 60 years of research on vasoactive peptides. The cardiovascular homeostasis derives from a balance of two systems, the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). The biologically active effector entity of RAS is angiotensin receptor-1 (AT-1R), and that of KKS is bradykinin B2 receptor (B2R). The first mediates vasoconstriction, the second is the most potent and efficient vasodilator. Thanks to its complex and multi-functional mechanism of action, involving nitric oxide (NO), prostacyclin and endothelial hyperpolarizing factor (EDHF). B2R is instrumental for the supply of blood, oxygen and nutrition to tissues. KKS is present on the vascular endothelium and functions as an autacoid playing major roles in cardiovascular diseases (CVDs) and diabetes. KKS exerts a paramount role in the prevention of thrombosis and atherosclerosis. Such knowledge emphasizes the already prominent value of the ACE-inhibitors (ACEIs) for the treatment of CVDs and diabetes. Indeed, the ACEIs, thanks to their double action (block of the RAS and potentiation of the KKS) are the ideal agents for a rational treatment of these diseases. PMID:26408609

  7. Antimicrobial peptides in annelids

    Directory of Open Access Journals (Sweden)

    A Tasiemski

    2008-06-01

    Full Text Available Gene encoded antimicrobial peptides (AMPs are widely distributed among living organisms including plants, invertebrates and vertebrates. They constitute important effectors of the innate immune response by exerting multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis and immune induction. In invertebrates, most of the data describe the characterization and/or the function of AMPs in the numerically and economically most representative group which are arthropods. Annelids are among the first coelomates and are therefore of special phylogenetic interest. Compared to other invertebrate groups, data on annelid’s immunity reveal heavier emphasis on the cellular than on the humoral response suggesting that immune defense of annelids seems to be principally developed as cellular immunity.This paper gives an overview of the variety of AMPs identified in the three classes of annelids, i.e. polychaetes, oligochaetes and achaetes. Their functions, when they have been studied, in the humoral or cellular response of annelids are also mentioned.

  8. Antimicrobial peptides in crustaceans

    Directory of Open Access Journals (Sweden)

    RD Rosa

    2010-11-01

    Full Text Available Crustaceans are a large and diverse invertebrate animal group that mounts a complex and efficient innate immune response against a variety of microorganisms. The crustacean immune system is primarily related to cellular responses and the production and release of important immune effectors into the hemolymph. Antimicrobial proteins and/or peptides (AMPs are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, 15 distinct AMP families are currently recognized, although the great majority (14 families comes from members of the order Decapoda. Crustacean AMPs are generally cationic, gene-encoded molecules that are mainly produced by circulating immune-competent cells (hemocytes or are derived from unrelated proteins primarily involved in other biological functions. In this review, we tentatively classified the crustacean AMPs into four main groups based on their amino acid composition, structural features and multi-functionality. We also attempted to summarize the current knowledge on their implication both in an efficient response to microbial infections and in crustacean survival.

  9. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

  10. Collagen-like antimicrobial peptides.

    Science.gov (United States)

    Masuda, Ryo; Kudo, Masakazu; Dazai, Yui; Mima, Takehiko; Koide, Takaki

    2016-11-01

    Combinatorial library composed of rigid rod-like peptides with a triple-helical scaffold was constructed. The component peptides were designed to have various combinations of basic and neutral (or hydrophobic) amino acid residues based on collagen-like (Gly-Pro-Yaa)-repeating sequences, inspired from the basic and amphiphilic nature of naturally occurring antimicrobial peptides. Screening of the peptide pools resulted in identification of antimicrobial peptides. A structure-activity relationship study revealed that the position of Arg-cluster at N-terminus and cystine knots at C-terminus in the triple helix significantly contributed to the antimicrobial activity. The most potent peptide RO-A showed activity against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. In addition, Escherichia coli exposed to RO-A resulted in abnormal elongation of the cells. RO-A was also shown to have remarkable stability in human serum and low cytotoxicity to mammalian cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 453-459, 2016. PMID:27271210

  11. Automated solid-phase peptide synthesis to obtain therapeutic peptides

    Directory of Open Access Journals (Sweden)

    Veronika Mäde

    2014-05-01

    Full Text Available The great versatility and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last years. In order to increase the drugability of these frequently unstable and rapidly cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide synthesis (SPPS offers a suitable technology to produce chemically engineered peptides. This review concentrates on the application of SPPS by Fmoc/t-Bu protecting-group strategy, which is most commonly used. Critical issues and suggestions for the synthesis are covered. The development of automated methods from conventional to essentially improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGylation are described as covalent conjugation methods, which can be applied by a combination of automated and manual synthesis approaches. The synthesis and application of SPPS is described for neuropeptide Y receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated synthesis technologies.

  12. Mycorrhizal-induced calmodulin mediated changes in antioxidant enzymes and growth response of drought-stressed trifoliate orange

    Directory of Open Access Journals (Sweden)

    Yong-Ming eHuang

    2014-12-01

    Full Text Available Trifoliate orange [Poncirus trifoliata (L Raf.] is considered highly arbuscular mycorrhizal (AM dependent for growth responses through a series of signal transductions in form of various physiological responses. The proposed study was carried out to evaluate the effect of an AM fungus (Funneliformis mosseae on growth, antioxidant enzyme (catalase, CAT; superoxide dismutase, SOD activities, leaf relative water content (RWC, calmodulin (CaM, superoxide anion (O2•− and hydrogen peroxide (H2O2 concentrations in leaves of the plants exposed to both well-watered (WW and drought stress (DS conditions. A 58-day of DS significantly decreased mycorrhizal colonization by 60% than WW. Compared to non-AM seedlings, AM seedlings displayed significantly higher shoot morphological properties (plant height, stem diameter and leaf number, biomass production (shoot and root fresh weight and leaf RWC, regardless of soil water status. AM inoculation significantly increased CaM and soluble protein concentrations and CAT activity, and significantly decreased O2•− and H2O2 concentration under both WW and DS conditions. The AM seedlings also exhibited significantly higher Cu/Zn-SOD and Mn-SOD activities than the non-AM seedlings under DS but not under WW, which are triggered by higher CaM levels in AM plants on the basis of correlation studies. Further, the negative correlation of Cu/Zn-SOD and Mn-SOD activities with O2•− and H2O2 concentration showed the DS-induced ROS scavenging ability of CaM mediated SODs under mycorrhization. Our results demonstrated that AM-inoculation elevated the synthesis of CaM in leaves and up-regulated activities of the antioxidant enzymes, thereby, repairing the possible oxidative damage to plants by lowering the ROS accumulation under DS condition.

  13. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.

  14. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    Science.gov (United States)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  15. Abscisic acid activates a Ca2+-calmodulin-stimulated protein kinase involved in antioxidant defense in maize leaves

    Institute of Scientific and Technical Information of China (English)

    Shucheng Xu

    2010-01-01

     The role of a calcium-dependent and calmodulin(CaM)stimulated protein kinase in abscisic acid(ABA)-induced antioxidant defense was determined in leaves of maize (Zea mays).In-gel kinase assays showed that treatments with ABA or H2O2 induced the activation of a 49-kDa protein kinase and a 52-kDa protein kinase significantly.Furthermore,we showed that the 52-kDa protein kinase has the characteristics of CaM-stimulating activity and is sensitive to calcium-CaM-dependent protein kinase Ⅱ (CaMK Ⅱ)inhibitor KN-93 or CaM antagonist W-7.Treatments with ABA or H2O2 not only induced the acti vation of the 52-kDa protein kinase,but also enhanced the total activities of the antioxidant enzymes,including catalase,ascorbate peroxidase,glutathione reductase,and superoxide dismutase.Such enhancements were blocked by pretreatment with a CaMK inhibitor and a reactive oxygen species(ROS)inhibitor or scavenger.Pretreatment with the CaMK inhibitor also substantially arrested the ABA-induced H2O2 production.Kinase activity enhancements induced by ABA were attenuated by pretreatment with an ROS inhibitor or scavenger.These results suggest that the 52-kDa CaMK is involved in ABA-induced antioxidant defense and that cross-talk between CaMK and H2O2 plays a pivotal role in ABA signaling.We infer that CaMK acts both upstream and downstream of H2O2,but mainly acts between ABA and H2O2 in ABA-induced antioxidant-defensive signaling.

  16. Nicotine reward and affective nicotine withdrawal signs are attenuated in calcium/calmodulin-dependent protein kinase IV knockout mice.

    Directory of Open Access Journals (Sweden)

    Kia J Jackson

    Full Text Available The influx of Ca(2+ through calcium-permeable nicotinic acetylcholine receptors (nAChRs leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB, which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (-/- mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV -/- mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.

  17. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  18. Involvement of calmodulin in regulation of primary root elongation by N-3-oxo-hexanoyl homoserine lactone in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Qian eZhao

    2015-01-01

    Full Text Available Many bacteria use signal molecules of low molecular weight to monitor their local population density and to coordinate their collective behavior in a process called quorum sensing (QS. N-acyl-homoserine lactones (AHLs are the primary QS signals among Gram-negative bacteria. AHL-mediated QS plays an essential role in diverse bacterial physiological processes. Recent evidence shows that plants are able to sense bacterial AHLs and respond to them appropriately. However, little is known about the mechanism by which plants perceive and transduce the bacterial AHLs within cells. In this study, we found that the stimulatory effect of N-3-oxo-hexanoyl homoserine lactone (3OC6-HSL on primary root elongation of Arabidopsis was abolished by the calmodulin (CaM antagonists N-(6-aminohexyl-5-chloro-1-naphthalene sulfonamide (W-7 and trifluoperazine (TFP. Western-blot and ELISA analysis revealed that the concentration of CaM protein in Arabidopsis roots increased after treatment with 1 μM 3OC6-HSL. Results from quantitative RT-PCR demonstrated that the transcription of all nine CaM genes in Arabidopsis genome was up-regulated in the plants treated with 3OC6-HSL. The loss-of-function mutants of each AtCaM gene (AtCaM1-9 were insensitive to 3OC6-HSL-stimulation of primary root elongation. On the other hand, the genetic evidence showed that CaM may not participates the inhibition of primary root length caused by application of long-chained AHLs such as C10-HSL and C12-HSL. Nevertheless, our results suggest that CaM is involved in the bacterial 3OC6-HSL signaling in plant cells. These data offer new insight into the mechanism of plant response to bacterial QS signals.

  19. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    International Nuclear Information System (INIS)

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca2+ and this interaction may be important for its invasion into animal cells

  20. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  1. Regulation of plant immunity through ubiquitin-mediated modulation of Ca(2+) -calmodulin-AtSR1/CAMTA3 signaling.

    Science.gov (United States)

    Zhang, Lei; Du, Liqun; Shen, Chenjia; Yang, Yanjun; Poovaiah, B W

    2014-04-01

    Transient changes in intracellular Ca(2+) concentration are essential signals for activation of plant immunity. It has also been reported that Ca(2+) signals suppress salicylic acid-mediated plant defense through AtSR1/CAMTA3, a member of the Ca(2+) /calmodulin-regulated transcription factor family that is conserved in multicellular eukaryotes. How plants overcome this negative regulation to mount an effective defense response during a stage of intracellular Ca(2+) surge is unclear. Here we report the identification and functional characterization of an important component of ubiquitin ligase, and the associated AtSR1 turnover. The AtSR1 interaction protein 1 (SR1IP1) was identified by CytoTrap two-hybrid screening. The loss-of-function mutant of SR1IP1 is more susceptible to bacterial pathogens, and over-expression of SR1IP1 confers enhanced resistance, indicating that SR1IP1 acts as a positive regulator of plant defense. SR1IP1 and AtSR1 act in the same signaling pathway to regulate plant immunity. SR1IP1 contains the structural features of a substrate adaptor in cullin 3-based E3 ubiquitin ligase, and was shown to serve as a substrate adaptor that recruits AtSR1 for ubiquitination and degradation when plants are challenged with pathogens. Hence, SR1IP1 positively regulates plant immunity by removing the defense suppressor AtSR1. These findings provide a mechanistic insight into how Ca(2+) -mediated actions are coordinated to achieve effective plant immunity. PMID:24528504

  2. Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform1[w

    Science.gov (United States)

    Turner, William L.; Waller, Jeffrey C.; Vanderbeld, Barb; Snedden, Wayne A.

    2004-01-01

    NAD kinase (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca2+-dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaM-dependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (KmNAD = 0.20 mM, KmMg2+−ATP = 0.17 mM). The enzyme was fully activated by conserved CaM (S0.5 = 2.2 nm) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins. Possible roles for NADKs in plants are discussed in light of our observations. PMID:15247403

  3. Approaches to the assignment of {sup 19}F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Kitevski-LeBlanc, Julianne L.; Evanics, Ferenc; Scott Prosser, R., E-mail: scott.prosser@utoronto.c [University of Toronto, Department of Chemistry (Canada)

    2010-06-15

    Traditional single site replacement mutations (in this case, phenylalanine to tyrosine) were compared with methods which exclusively employ {sup 15}N and {sup 19}F-edited two- and three-dimensional NMR experiments for purposes of assigning {sup 19}F NMR resonances from calmodulin (CaM), biosynthetically labeled with 3-fluorophenylalanine (3-FPhe). The global substitution of 3-FPhe for native phenylalanine was tolerated in CaM as evidenced by a comparison of {sup 1}H-{sup 15}N HSQC spectra and calcium binding assays in the presence and absence of 3-FPhe. The {sup 19}F NMR spectrum reveals six resolved resonances, one of which integrates to three 3-FPhe species, making for a total of eight fluorophenylalanines. Single phenylalanine to tyrosine mutants of five phenylalanine positions resulted in {sup 19}F NMR spectra with significant chemical shift perturbations of the remaining resonances, and provided only a single definitive assignment. Although {sup 1}H-{sup 19}F heteronucleclear NOEs proved weak, {sup 19}F-edited {sup 1}H-{sup 1}H NOESY connectivities were relatively easy to establish by making use of the {sup 3}J{sub FH} coupling between the fluorine nucleus and the adjacent fluorophenylalanine {delta} proton. {sup 19}F-edited NOESY connectivities between the {delta} protons and {alpha} and {beta} nuclei in addition to {sup 15}N-edited {sup 1}H, {sup 1}H NOESY crosspeaks proved sufficient to assign 4 of 8 {sup 19}F resonances. Controlled cleavage of the protein into two fragments using trypsin, and a repetition of the above 2D and 3D techniques resulted in unambiguous assignments of all 8 {sup 19}F NMR resonances. Our studies suggest that {sup 19}F-edited NOESY NMR spectra are generally adequate for complete assignment without the need to resort to mutational analysis.

  4. Peptides and Food Intake

    Directory of Open Access Journals (Sweden)

    Carmen Sobrino Crespo

    2014-04-01

    Full Text Available Nutrients created by the digestion of food are proposed to active G protein coupled receptors on the luminal side of enteroendocrine cells e.g. the L-cell. This stimulates the release of gut hormones. Hormones released from the gut and adipose tissue play an important rol in the regulation of food intake and energy expenditure (1.Many circulating signals, including gut hormones, can influence the activity of the arcuate nucleus (ARC neurons directly, after passing across the median eminence. The ARC is adjacent to the median eminence, a circumventricular organ with fenestrated capillaries and hence an incomplete blood-brain barrier (2. The ARC of the hypothalamus is believed to play a crucial role in the regulation of food intake and energy homeostasis. The ARC contains two populations of neurons with opposing effect on food intake (3. Medially located orexigenic neurons (i.e those stimulating appetite express neuropeptide Y (NPY and agouti-related protein (AgRP (4-5. Anorexigenic neurons (i.e. those inhibiting appetite in the lateral ARC express alpha-melanocyte stimulating hormone (α-MSH derived from pro-opiomelanocortin (POMC and cocaine and amphetamine-regulated transcript (CART (6. The balance between activities of these neuronal circuits is critical to body weight regulation.In contrast, other peripheral signals influence the hypothalamus indirectly via afferent neuronal pathway and brainstem circuits. In this context gastrointestinal’s vagal afferents are activated by mechanoreceptors and chemoreceptors, and converge in the nucleus of the tractus solitaries (NTS of the brainstem. Neuronal projections from the NTS, in turn, carry signals to the hypotalamus (1, 7. Gut hormones also alter the activity of the ascending vagal pathway from the gut to the brainstem. In the cases of ghrelin and Peptide tyrosine tyrosine (PYY, there are evidences for both to have a direct action on the arcuate nucleus and an action via the vagus nerve a

  5. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongliang; Hong, Da Hye; Kim, Han Sol; Kim, Hye Won [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of); Jung, Won-Kyo [Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan 608-737 (Korea, Republic of); Na, Sung Hun [Institute of Medical Sciences, Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, 200-701 (Korea, Republic of); Jung, In Duk; Park, Yeong-Min [Department of Immunology, Lab of Dendritic Cell Differentiation and Regulation, College of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Choi, Il-Whan, E-mail: cihima@inje.ac.kr [Department of Microbiology, Inje University College of Medicine, Busan, 614-735 (Korea, Republic of); Park, Won Sun, E-mail: parkws@kangwon.ac.kr [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of)

    2015-06-15

    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

  6. Molecular characterisation of a calmodulin gene, VcCaM1, that is differentially expressed under aluminium stress in highbush blueberry.

    Science.gov (United States)

    Inostroza-Blancheteau, C; Aquea, F; Loyola, R; Slovin, J; Josway, S; Rengel, Z; Reyes-Díaz, M; Alberdi, M; Arce-Johnson, P

    2013-11-01

    Calmodulin (CaM), a small acidic protein, is one of the best characterised Ca(2+) sensors in eukaryotes. This Ca(2+) -regulated protein plays a critical role in decoding and transducing environmental stress signals by activating specific targets. Many environmental stresses elicit changes in intracellular Ca(2+) activity that could initiate adaptive responses under adverse conditions. We report the first molecular cloning and characterisation of a calmodulin gene, VcCaM1 (Vaccinium corymbosum Calmodulin 1), in the woody shrub, highbush blueberry. VcCaM1 was first identified as VCAL19, a gene induced by aluminium stress in V. corymbosum L. A full-length cDNA of VcCaM1 containing a 766-bp open reading frame (ORF) encoding 149 amino acids was cloned from root RNA. The sequence encodes four Ca(2+) -binding motifs (EF-hands) and shows high similarity (99%) with the isoform CaM 201 of Daucus carota. Expression analyses showed that following Al treatment, VcCaM1 message level decreased in roots of Brigitta, an Al-resistant cultivar, and after 48 h, was lower than in Bluegold, an Al-sensitive cultivar. VcCAM1 message also decreased in leaves of both cultivars within 2 h of treatment. Message levels in leaves then increased by 24 h to control levels in Brigitta, but not in Bluegold, but then decreased again by 48 h. In conclusion, VcCaM1 does not appear to be directly involved in Al resistance, but may be involved in improved plant performance under Al toxicity conditions through regulation of Ca(2+) homeostasis and antioxidant systems in leaves.

  7. Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

    Science.gov (United States)

    Lou, Jie; Zhao, Dan; Zhang, Ling-Ling; Song, Shu-Ying; Li, Yan-Chao; Sun, Fei; Ding, Xiao-Qing; Yu, Chang-Jiang; Li, Yuan-Yuan; Liu, Mei-Tong; Dong, Chang-Jiang; Ji, Yong; Li, Hongliang; Chu, Wenfeng; Zhang, Zhi-Ren

    2016-09-01

    The role of type III transforming growth factor-β receptor (TβRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TβRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TβRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TβRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TβRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TβRIII in vitro. Cardiac-specific transgenic expression of TβRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TβRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TβRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TβRIII in vivo. Our data suggest that TβRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of β-arrestin2 with both TβRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TβRIII expression results in cardiac hypertrophy through β-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-β and β-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac

  8. Targeting cell migration and the endoplasmic reticulum stress response with calmodulin antagonists: a clinically tested small molecule phenocopy of SEC62 gene silencing in human tumor cells

    International Nuclear Information System (INIS)

    Tumor cells benefit from their ability to avoid apoptosis and invade other tissues. The endoplasmic reticulum (ER) membrane protein Sec62 is a key player in these processes. Sec62 is essential for cell migration and protects tumor cells against thapsigargin-induced ER stress, which are both linked to cytosolic Ca2+. SEC62 silencing leads to elevated cytosolic Ca2+ and increased ER Ca2+ leakage after thapsigargin treatment. Sec62 protein levels are significantly increased in different tumors, including prostate, lung and thyroid cancer. In lung cancer, the influence of Sec62 protein levels on patient survival was analyzed using the Kaplan-Meier method and log-rank test. To elucidate the underlying pathophysiological functions of Sec62, Ca2+ imaging techniques, real-time cell analysis and cell migration assays were performed. The effects of treatment with the calmodulin antagonists, trifluoperazine (TFP) and ophiobolin A, on cellular Ca2+ homeostasis, cell growth and cell migration were compared with the effects of siRNA-mediated Sec62 depletion or the expression of a mutated SEC62 variant in vitro. Using Biacore analysis we examined the Ca2+-sensitive interaction of Sec62 with the Sec61 complex. Sec62 overproduction significantly correlated with reduced patient survival. Therefore, Sec62 is not only a predictive marker for this type of tumor, but also an interesting therapeutic target. The present study suggests a regulatory function for Sec62 in the major Ca2+ leakage channel in the ER, Sec61, by a direct and Ca2+-sensitive interaction. A Ca2+-binding motif in Sec62 is essential for its molecular function. Treatment of cells with calmodulin antagonists mimicked Sec62 depletion by inhibiting cell migration and rendering the cells sensitive to thapsigargin treatment. Targeting tumors that overproduce Sec62 with calmodulin antagonists in combination with targeted thapsigargin analogues may offer novel personalized therapeutic options

  9. Peptides: A new class of anticancer drugs

    Directory of Open Access Journals (Sweden)

    Ryszard Smolarczyk

    2009-07-01

    Full Text Available Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides, which bind specifically to precise molecular targets located on the surface of tumor cells. Antibacterial peptides bind to both cell and mitochondrial membranes. Some of these peptides attach to the cell membrane, resulting in its disorganization. Other antibacterial peptides penetrate cancer cells without causing cell membrane damage, but they disrupt mitochondrial membranes. Thanks to phage and aptamer libraries, it has become possible to obtain synthetic peptides blocking or activating some target proteins found in cancer cells as well as in cells forming the tumor environment. These synthetic peptides can feature anti-angiogenic properties, block enzymes indispensable for sustained tumor growth, and reduce tumor ability to metastasize. In this review the properties of peptides belonging to both categories are discussed and attempts of their application for therapeutic purposes are outlined.

  10. Perspectives and Peptides of the Next Generation

    Science.gov (United States)

    Brogden, Kim A.

    Shortly after their discovery, antimicrobial peptides from prokaryotes and eukaryotes were recognized as the next potential generation of pharmaceuticals to treat antibiotic-resistant bacterial infections and septic shock, to preserve food, or to sanitize surfaces. Initial research focused on identifying the spectrum of antimicrobial agents, determining the range of antimicrobial activities against bacterial, fungal, and viral pathogens, and assessing the antimicrobial activity of synthetic peptides versus their natural counterparts. Subsequent research then focused on the mechanisms of antimicrobial peptide activity in model membrane systems not only to identify the mechanisms of antimicrobial peptide activity in microorganisms but also to discern differences in cytotoxicity for prokaryotic and eukaryotic cells. Recent, contemporary work now focuses on current and future efforts to construct hybrid peptides, peptide congeners, stabilized peptides, peptide conjugates, and immobilized peptides for unique and specific applications to control the growth of microorganisms in vitro and in vivo.

  11. Exploration of the Medicinal Peptide Space.

    Science.gov (United States)

    Gevaert, Bert; Stalmans, Sofie; Wynendaele, Evelien; Taevernier, Lien; Bracke, Nathalie; D'Hondt, Matthias; De Spiegeleer, Bart

    2016-01-01

    The chemical properties of peptide medicines, known as the 'medicinal peptide space' is considered a multi-dimensional subset of the global peptide space, where each dimension represents a chemical descriptor. These descriptors can be linked to biofunctional, medicinal properties to varying degrees. Knowledge of this space can increase the efficiency of the peptide-drug discovery and development process, as well as advance our understanding and classification of peptide medicines. For 245 peptide drugs, already available on the market or in clinical development, multivariate dataexploration was performed using peptide relevant physicochemical descriptors, their specific peptidedrug target and their clinical use. Our retrospective analysis indicates that clusters in the medicinal peptide space are located in a relatively narrow range of the physicochemical space: dense and empty regions were found, which can be explored for the discovery of novel peptide drugs. PMID:26876881

  12. Regulation of Voltage-Gated Ca2+ Currents by Ca2+/Calmodulin-dependent Protein Kinase II in Resting Sensory Neurons

    OpenAIRE

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-en; Hogan, Quinn H

    2014-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca2+ channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca2+ currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced...

  13. Ca2+/Calmodulin Kinase Kinase α Is Dispensable for Brain Development but Is Required for Distinct Memories in Male, though Not in Female, Mice▿

    OpenAIRE

    Mizuno, Keiko; Ris, Laurence; Sánchez-Capelo, Amelia; Godaux, Emile; Giese, K. Peter

    2006-01-01

    In neurons, the Ca2+/calmodulin (CaM) kinase cascade transduces Ca2+ signaling into gene transcription. The CaM kinase cascade is known to be important for brain development as well as memory formation in adult brain, although the functions of some cascade members remain unknown. Here we have generated null and hypomorphic mutants to study the physiological role of CaM kinase kinase α (CaMKKα), which phosphorylates and activates both CaM kinase I (CaMKI) and CaMKIV, the output kinases of the ...

  14. Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

    OpenAIRE

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M.

    2009-01-01

    Voltage-activated Cav1.2 calcium channels require association of the pore-forming α1C subunit with accessory Cavβ and α2δ subunits. Binding of a single calmodulin (CaM) to α1C supports Ca2+-dependent inactivation (CDI). The human Cav1.2 channel is silent in the absence of Cavβ and/or α2δ. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Cavβ. Here we discovered that CaMex and its Ca2+...

  15. The Activation Effect of Hainantoxin-I, a Peptide Toxin from the Chinese Spider, Ornithoctonus hainana, on Intermediate-Conductance Ca2+-Activated K+ Channels

    Directory of Open Access Journals (Sweden)

    Pengfei Huang

    2014-08-01

    Full Text Available Intermediate-conductance Ca2+-activated K+ (IK channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.

  16. Peptide Vaccine: Progress and Challenges

    Directory of Open Access Journals (Sweden)

    Weidang Li

    2014-07-01

    Full Text Available Conventional vaccine strategies have been highly efficacious for several decades in reducing mortality and morbidity due to infectious diseases. The bane of conventional vaccines, such as those that include whole organisms or large proteins, appear to be the inclusion of unnecessary antigenic load that, not only contributes little to the protective immune response, but complicates the situation by inducing allergenic and/or reactogenic responses. Peptide vaccines are an attractive alternative strategy that relies on usage of short peptide fragments to engineer the induction of highly targeted immune responses, consequently avoiding allergenic and/or reactogenic sequences. Conversely, peptide vaccines used in isolation are often weakly immunogenic and require particulate carriers for delivery and adjuvanting. In this article, we discuss the specific advantages and considerations in targeted induction of immune responses by peptide vaccines and progresses in the development of such vaccines against various diseases. Additionally, we also discuss the development of particulate carrier strategies and the inherent challenges with regard to safety when combining such technologies with peptide vaccines.

  17. Recent development of peptide self-assembly

    Institute of Scientific and Technical Information of China (English)

    Xiubo Zhao; Fang Pan; Jian R. Lu

    2008-01-01

    Amino acids are the building blocks to build peptides and proteins. Recent development in peptide synthesis has however enabled us to mimic this natural process by preparing various long and short peptides possessing different conformations and biological functions. The self-assembly of short designed peptides into molecular nanostructures is becoming a growing interest in nanobiotechnology. Self-assembled peptides exhibit several attractive features for applications in tissue regeneration, drug delivery, biological surface engineering as well as in food science, cosmetic industry and antibiotics. The aim of this review is to introduce the readers to a number of representative studies on peptide self-assembly.

  18. Antiviral active peptide from oyster

    Science.gov (United States)

    Zeng, Mingyong; Cui, Wenxuan; Zhao, Yuanhui; Liu, Zunying; Dong, Shiyuan; Guo, Yao

    2008-08-01

    An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster ( Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10 5 kDa, 5 1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10 5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

  19. Antiviral active peptide from oyster

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster (Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10-5 kDa, 5-1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10?5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

  20. Radioactive labelling of peptidic hormones

    International Nuclear Information System (INIS)

    The labelling of peptidic hormones requires stability, specificity and sensitivity of the label. Introduction of a radioactive atome is one way to satisfy these criteria. Several processes have been described to prepare radioactive TRF: synthesis of the peptide with labelled aminoacids or introduction of the label into the hormone. In that approach, tritium can be substituted in the imidazole ring, via precursors activating the proper carbon. Monoiodo TRF leads essentially to tritium labelling of the 5 positions whereas monoazo TRF allows the preparation of 3H TRF labelled in the 2 positions. Di-substituted TRF leads to labelling into the 2 and 5 carbons. Labelled analogs of TRF can be prepared with labelled iodine; further developments of peptide labelling, will be presented. In particular, the homolytic scission of the C-iodine, bond by photochemical activation. The nascent carbon radical can be stabilized by a tritiated scavenger. This approach eliminates the use of heavy metal catalysts

  1. Peptide Antibiotics for ESKAPE Pathogens

    DEFF Research Database (Denmark)

    Thomsen, Thomas Thyge

    is considered poor compared to medicines for lifestyle diseases. According to the WHO we could be moving towards a post-antibiotic era in which previously treatable infections become fatal. Of special importance are multidrug resistant bacteria from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus...... a cecropin-mellitin hybrid peptide and proved effective in killing colistin resistant Gram-negative A. baumannii in vitro. The molecule was improved with regard to toxicity, as measured by hemolytic ability. Further, this peptide is capable of specifically killing non-growing cells of colistin resistant A...

  2. Peptides and the new endocrinology

    Science.gov (United States)

    Schwyzer, Robert

    1982-01-01

    The discovery of regulatory peptides common to the nervous and the endocrine systems (brain, gut, and skin) has brought about a revolution in our concepts of endocrinology and neurology. We are beginning to understand some of the complex interrelationships between soma and psyche that might, someday, be important for an integrated treatment of diseases. Examples of the actions of certain peptides in the periphery and in the central nervous system are given, and their biosynthesis and molecular anatomy as carriers for information are discussed.

  3. Novel Formulations for Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Ana Maria Carmona-Ribeiro

    2014-10-01

    Full Text Available Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy.

  4. An enhancer peptide for membrane-disrupting antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Zhang Hong

    2010-02-01

    Full Text Available Abstract Background NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4 by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn. Results In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against Staphylococcus aureus, whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P. Conclusions NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane.

  5. The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

    Science.gov (United States)

    Kool, Martijn J; van de Bree, Jolet E; Bodde, Hanna E; Elgersma, Ype; van Woerden, Geeske M

    2016-01-01

    Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2b(f/f) and Camk2b(T287A)), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b(-/-) mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion. PMID:27244486

  6. Overexpression of calmodulin-like (ShCML44) stress-responsive gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses.

    Science.gov (United States)

    Munir, Shoaib; Liu, Hui; Xing, Yali; Hussain, Saddam; Ouyang, Bo; Zhang, Yuyang; Li, Hanxia; Ye, Zhibiao

    2016-01-01

    Calmodulin-like (CML) proteins are important Ca(2+) sensors, which play significant role in mediating plant stress tolerance. In the present study, cold responsive calmodulin-like (ShCML44) gene was isolated from cold tolerant wild tomato (Solanum habrochaites), and functionally characterized. The ShCML44 was differentially expressed in all plant tissues including root, stem, leaf, flower and fruit, and was strongly up-regulated under cold, drought and salinity stresses along with plant growth hormones. Under cold stress, progressive increase in the expression of ShCML44 was observed particularly in cold-tolerant S. habrochaites. The ShCML44-overexpressed plants showed greater tolerance to cold, drought, and salinity stresses, and recorded higher germination and better seedling growth. Transgenic tomato plants demonstrated higher antioxidant enzymes activity, gas exchange and water retention capacity with lower malondialdehyde accumulation and membrane damage under cold and drought stresses compared to wild-type. Moreover, transgenic plants exhibited reduced reactive oxygen species and higher relative water contents under cold and drought stress, respectively. Greater stress tolerance of transgenic plants was further reflected by the up-/down-regulation of stress-related genes including SOD, GST, CAT, POD, LOX, PR and ERD. In crux, these results strengthen the molecular understanding of ShCML44 gene to improve the abiotic stress tolerance in tomato. PMID:27546315

  7. Strategic approaches to optimizing peptide ADME properties.

    Science.gov (United States)

    Di, Li

    2015-01-01

    Development of peptide drugs is challenging but also quite rewarding. Five blockbuster peptide drugs are currently on the market, and six new peptides received first marketing approval as new molecular entities in 2012. Although peptides only represent 2% of the drug market, the market is growing twice as quickly and might soon occupy a larger niche. Natural peptides typically have poor absorption, distribution, metabolism, and excretion (ADME) properties with rapid clearance, short half-life, low permeability, and sometimes low solubility. Strategies have been developed to improve peptide drugability through enhancing permeability, reducing proteolysis and renal clearance, and prolonging half-life. In vivo, in vitro, and in silico tools are available to evaluate ADME properties of peptides, and structural modification strategies are in place to improve peptide developability. PMID:25366889

  8. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  9. Lipoxygenase inhibitor peptides and their use

    OpenAIRE

    Schurink, M.; Boeriu, C.G.; Berkel, van, A.M.; Wichers, H J

    2006-01-01

    The present invention is in the field of enzyme inhibition. In particular it relates to peptide inhibitors for lipoxygenases. The lipoxygenase peptide inhibitors of have the potential to be used as therapeutic drugs as well as food preservatives.

  10. Natriuretic peptides in cardiometabolic regulation and disease

    DEFF Research Database (Denmark)

    Zois, Nora Elisabeth; Bartels, Emil Daniel; Hunter, Ingrid;

    2014-01-01

    decade. Dysregulation of the natriuretic peptide system has been associated with obesity, glucose intolerance, type 2 diabetes mellitus, and essential hypertension. Moreover, the natriuretic peptides have been implicated in the protection against atherosclerosis, thrombosis, and myocardial ischaemia. All...

  11. The solution structures of two soybean calmodulin isoforms provide a structural basis for their selective target activation properties.

    Science.gov (United States)

    Ishida, Hiroaki; Huang, Hao; Yamniuk, Aaron P; Takaya, Yoshiaki; Vogel, Hans J

    2008-05-23

    The intracellular calcium ion is one of the most important secondary messengers in eukaryotic cells. Ca(2+) signals are translated into physiological responses by EF-hand calcium-binding proteins such as calmodulin (CaM). Multiple CaM isoforms occur in plant cells, whereas only a single CaM protein is found in animals. Soybean CaM isoform 1 (sCaM1) shares 90% amino acid sequence identity with animal CaM (aCaM), whereas sCaM4 is only 78% identical. These two sCaM isoforms have distinct target-enzyme activation properties and physiological functions. sCaM4 is highly expressed during the self-defense reaction of the plant and activates the enzyme nitric-oxide synthase (NOS), whereas sCaM1 is incapable of activating NOS. The mechanism of selective target activation by plant CaM isoforms is poorly understood. We have determined high resolution NMR solution structures of Ca(2+)-sCaM1 and -sCaM4. These were compared with previously determined Ca(2+)-aCaM structures. For the N-lobe of the protein, the solution structures of Ca(2+)-sCaM1, -sCaM4, and -aCaM all closely resemble each other. However, despite the high sequence identity with aCaM, the C-lobe of Ca(2+)-sCaM1 has a more open conformation and consequently a larger hydrophobic target-protein binding pocket than Ca(2+)-aCaM or -sCaM4, the presence of which was further confirmed through biophysical measurements. The single Val-144 --> Met substitution in the C-lobe of Ca(2+)-sCaM1, which restores its ability to activate NOS, alters the structure of the C-lobe to a more closed conformation resembling Ca(2+)-aCaM and -sCaM4. The relationships between the structural differences in the two Ca(2+)-sCaM isoforms and their selective target activation properties are discussed. PMID:18347016

  12. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  13. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin.

    Science.gov (United States)

    Springer, Tzvia I; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L

    2014-10-10

    Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD's β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD's β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM.

  14. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93 calmodulin cDNA using computational tools

    Directory of Open Access Journals (Sweden)

    Kassim Amelia

    2015-01-01

    Full Text Available Background: Common bean (Phaseolus vulgaris L. is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM gene cDNA and its deduced protein (amino acids sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is

  15. Purification, structure and function of bioactive peptides

    OpenAIRE

    Eriste, Elo

    2004-01-01

    Peptides are vitally important molecules and many evoke cellular responses. The completion of several genome sequencing projects has revealed a number of new genes. However, as functional peptides often contain posttranslational modifications and/or occur at various lengths, it is of great importance to detect, purify and characterize novel bioactive peptides. To achieve these goals, new methods for peptide detection, isolation and functional characterization have to be d...

  16. Development and use of engineered peptide deformylase in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia

    2012-01-01

    Deze thesis beschrijft het onderzoek naar potentieel van het gebruik van het peptide deformylase (PDF) in chemo enzymatische peptide synthese. PDF is geschikt voor selective N terminale deformylatie van bepaalde N-formyl-peptides zonder gelijktijdige hydrolyse van de peptide binding. Door de uitdagi

  17. Interpreting peptide mass spectra by VEMS

    DEFF Research Database (Denmark)

    Mathiesen, Rune; Lundsgaard, M.; Welinder, Karen G.;

    2003-01-01

    of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares...

  18. Synthetic Procedures for Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  19. Characterization of synthetic peptides by mass spectrometry

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Mirza, Osman Asghar; Højrup, Peter;

    2015-01-01

    Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI-TOF-MS an...

  20. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  1. Peptides and metallic nanoparticles for biomedical applications.

    NARCIS (Netherlands)

    Kogan, M.J.; Olmedo, I.; Hosta, L.; Guerrero, A.R.; Cruz Ricondo, L.J.; Albericio, F.

    2007-01-01

    In this review, we describe the contribution of peptides to the biomedical applications of metallic nanoparticles. We also discuss strategies for the preparation of peptide-nanoparticle conjugates and the synthesis of the peptides and metallic nanoparticles. An overview of the techniques used for th

  2. Diversity of wheat anti-microbial peptides.

    Science.gov (United States)

    Egorov, Tsezi A; Odintsova, Tatyana I; Pukhalsky, Vitaliy A; Grishin, Eugene V

    2005-11-01

    From seeds of Triticum kiharae Dorof. et Migusch., 24 novel anti-microbial peptides were isolated and characterized by a combination of three-step HPLC (affinity, size-exclusion and reversed-phase) with matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and Edman degradation. Based on sequence similarity and cysteine motifs, partially sequenced peptides were assigned to 7 families: defensins, thionins, lipid-transfer proteins, hevein-like peptides, knottin-like peptides, glycine-rich peptides, and MBP-1 homologs. A novel subfamily of defensins consisting of 6 peptides and a new family of glycine-rich (8 peptides with different repeat motifs) were identified. Three 6-cysteine knottin-like peptides represented by N- and C-terminally truncated variants revealed no sequence homology to any known plant anti-microbial peptides. A new 8-cysteine hevein-like peptide and three 4-cysteine peptides homologous to MBP-1 from maize were isolated. This is the first communication on the occurrence of nearly all families of plant anti-microbial peptides in a single species. PMID:16269343

  3. Toxins and antimicrobial peptides: interactions with membranes

    Science.gov (United States)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

  4. Bioactive peptides in dairy products

    Directory of Open Access Journals (Sweden)

    Donata Marletta

    2010-01-01

    Full Text Available Bioactive peptides are specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health. Most of the biological activities are encrypted within the primary sequence of the native protein and can be released by enzymatic hydrolysis and proteolysis or by food processing. Milk is a rich source of bioactive peptides which may contribute to regulate the nervous, gastrointestinal and cardiovascular systems as well as the immune system, confirming the added value of dairy products that, in certain cases, can be considered functional foods. The main biological activities of these peptides and their bioavailability in dairy products are reviewed. The natural concentration of these biomolecules is quite low and, to date one of the main goals has been to realize products enriched with bioactive peptides that have beneficial effects on human health and proven safety. Even though several health-enhancing products have already been launched and their integration in the diet could help in the prevention of chronic diseases such as hypertension, cancer and osteoporosis, more clinical trials are required in order to develop a deeper understanding of the activity of biopeptides on the human physiological mechanisms and also to assess the efficacy of their effects in a long term view. New scientific data are also needed to support their commercialisation in compliance with current regulations.

  5. Novel and Convenient Method for the Preparation of Phosphonate Peptides and Phosphonamidate Peptides

    Institute of Scientific and Technical Information of China (English)

    XU Jia-Xi; FU Nan-Yan; GAO Yuan-He; ZHNAG Qi-Han; DUAN Li-Fang

    2003-01-01

    @@ Phosphonate and phosphonamidate peptides are phosphorus analogues of natural peptides. They have been great used as stable mimetics of tetrahedral transition states as enzyme inhibitors and as haptens for catalytic antibody research in recent years. Although several methods are available for the preparation of phosphonate peptides and phosphonamidate peptides, all of them use phosphonic acid derivatives as starting materials. The overall yields from the synthesis of phosphonic acid derivatives to desired peptides are not satisfactory in most cases.

  6. Fabrication of Odor Sensor Using Peptide

    Science.gov (United States)

    Hotokebuchi, Yuta; Hayashi, Kenshi; Toko, Kiyoshi; Chen, Ronggang; Ikezaki, Hidekazu

    We report fabrication of an odor sensor using peptides. Peptides were designed to acquire the specific reception for a target odor molecule. Au surface of the sensor electrode was coated by the designed peptide using the method of self assembled monolayers (SAMs). Functionalized Au surfaces by the peptides were confirmed by ellipsometry and cyclic voltammetry. The odorants of vanillin, phenethyl alcohol and hexanol were discriminated by QCM sensor with the peptide surface. Moreover, we verified specific interaction between amino acid (Trp) and vanillin by fluorescence assay.

  7. Computer-Aided Design of Antimicrobial Peptides

    DEFF Research Database (Denmark)

    Fjell, Christopher D.; Hancock, Robert E.W.; Jenssen, Håvard

    2010-01-01

    chemical parameters with biological activities of the peptide, using statistical methods. In this review we will discuss two different in silico strategies of computer-aided antibacterial peptide design, a linear correlation model build as an extension of traditional principal component analysis (PCA......) and a non-linear artificial neural network model. Studies on structurally diverse peptides, have concluded that the PCA derived model are able to guide the antibacterial peptide design in a meaningful way, however requiring rather a high homology between the peptides in the test-set and the in silico...

  8. Peptide-enhanced oral delivery of therapeutic peptides and proteins

    DEFF Research Database (Denmark)

    Kristensen, Mie; Foged, Camilla; Berthelsen, Jens;

    2013-01-01

    Systemic therapy upon oral delivery of biologics, such as peptide and protein drugs is limited due to their large molecular size, their low enzymatic stability and their inability to cross the intestinal epithelium. Ways to overcome the epithelial barrier include the use of peptide-based excipients...... throughout the gastrointestinal (GI) tract, chemical stability is an inherent challenge when employing amino acid-based excipients for oral delivery, and multiple approaches have been investigated to improve this. The exact mechanisms of transepithelial translocation are discussed, and it is believed...... that CPP-mediated translocation involves transcytosis and/or direct translocation through the epithelial cells; whereas TJMP-mediated translocation is dependent on interaction with transmembrane or peripheral TJ proteins. This review focuses on the CPPs and the TJMPs currently employed as excipients...

  9. Towards the MHC-peptide combinatorics.

    Science.gov (United States)

    Kangueane, P; Sakharkar, M K; Kolatkar, P R; Ren, E C

    2001-05-01

    The exponentially increased sequence information on major histocompatibility complex (MHC) alleles points to the existence of a high degree of polymorphism within them. To understand the functional consequences of MHC alleles, 36 nonredundant MHC-peptide complexes in the protein data bank (PDB) were examined. Induced fit molecular recognition patterns such as those in MHC-peptide complexes are governed by numerous rules. The 36 complexes were clustered into 19 subgroups based on allele specificity and peptide length. The subgroups were further analyzed for identifying common features in MHC-peptide binding pattern. The four major observations made during the investigation were: (1) the positional preference of peptide residues defined by percentage burial upon complex formation is shown for all the 19 subgroups and the burial profiles within entries in a given subgroup are found to be similar; (2) in class I specific 8- and 9-mer peptides, the fourth residue is consistently solvent exposed, however this observation is not consistent in class I specific 10-mer peptides; (3) an anchor-shift in positional preference is observed towards the C terminal as the peptide length increases in class II specific peptides; and (4) peptide backbone atoms are proportionately dominant at the MHC-peptide interface.

  10. Natural and synthetic peptides with antifungal activity.

    Science.gov (United States)

    Ciociola, Tecla; Giovati, Laura; Conti, Stefania; Magliani, Walter; Santinoli, Claudia; Polonelli, Luciano

    2016-08-01

    In recent years, the increase of invasive fungal infections and the emergence of antifungal resistance stressed the need for new antifungal drugs. Peptides have shown to be good candidates for the development of alternative antimicrobial agents through high-throughput screening, and subsequent optimization according to a rational approach. This review presents a brief overview on antifungal natural peptides of different sources (animals, plants, micro-organisms), peptide fragments derived by proteolytic cleavage of precursor physiological proteins (cryptides), synthetic unnatural peptides and peptide derivatives. Antifungal peptides are schematically reported based on their structure, antifungal spectrum and reported effects. Natural or synthetic peptides and their modified derivatives may represent the basis for new compounds active against fungal infections. PMID:27502155

  11. Flourescent Peptide-Stabilized Silver-Nanoclusters

    DEFF Research Database (Denmark)

    Gregersen, Simon

    for instance small molecules, DNA oligomers, and proteins. Peptides are an intriguing class of biomolecular ligands, due to the large combinatorial space these provide. Furthermore, as peptides have a propensity to fold up into well-defined and somewhat rigid secondary structures, they may serve as excellent...... throughput dramatically with regards to discovery of novel ligands. Our approach employs Fmoc solid-phase peptide synthesis on a PEGA resin which allows for on-resin screening of peptide ligands which, in turn, removes the tedious and labor-intensive work-up of synthesized peptides. The method allows for on......-resin formation of peptide-stabilized Ag-NCs in a reversible manner, which makes identification of novel lead compound from combinatorial peptide libraries possible with a few simple steps. This resulted in the discovery of at least one promising candidate (P262) showing brighter emission, spectral homogeneity...

  12. 植物具IQ基序的钙调素结合蛋白的研究进展%Research Progress in Plant IQ Motif-containing Calmodulin-binding Proteins

    Institute of Scientific and Technical Information of China (English)

    田长恩; 周玉萍

    2013-01-01

    钙调素作为细胞内主要的Ca2+传感蛋白,通过与不同的钙调素结合蛋白的结合传递钙信号,调控细胞生理和生长发育过程.IQ基序(IQxxxRGxxxR,Pfam 00612)是少数几个钙调素与钙调素结合蛋白结合的结构域之一.植物具IQ基序的钙调素结合蛋白包括IQM、IQD、CAMTA、CNGC和myosin 5个家族及少数其它蛋白.该文综述了植物具IQ基序的钙调素结合蛋白的类型、结构特点和功能等方面的研究进展,并对今后的研究进行了展望.%As the primary intracellular calcium sensors,calmodulins regulate different cellular physiologic,growth and development processes by binding to different calmodulin-binding proteins.The IQ motif,IQxxxRGxxxR (Pfam 00612),is one of a few recognition motifs for calmodulins in calmodulin-binding proteins.As well as a few other proteins,5 major families,including IQM,IQD,CAMTA,CNGC and myosin family,have been found to possess the IQ motif(s) in plants.Here,we review the research progress in plant IQ motif-containing calmodulin-binding proteins,including their type,structural characteristics and function,and prospects for future study.

  13. Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

    Directory of Open Access Journals (Sweden)

    Gonzalez-Mendez Ricardo

    2011-07-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These

  14. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions

    DEFF Research Database (Denmark)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco;

    2016-01-01

    . In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited...... for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions...... determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide...

  15. Encapsulation of bioactive whey peptides in soy lecithin-derived nanoliposomes: Influence of peptide molecular weight.

    Science.gov (United States)

    Mohan, Aishwarya; McClements, David Julian; Udenigwe, Chibuike C

    2016-12-15

    Encapsulation of peptides can be used to enhance their stability, delivery and bioavailability. This study focused on the effect of the molecular weight range of whey peptides on their encapsulation within soy lecithin-derived nanoliposomes. Peptide molecular weight did not have a major impact on encapsulation efficiency or liposome size. However, it influenced peptide distribution amongst the surface, core, and bilayer regions of the liposomes, as determined by electrical charge (ζ-potential) and FTIR analysis. The liposome ζ-potential depended on peptide molecular weight, suggesting that the peptide charged groups were in different locations relative to the liposome surfaces. FTIR analysis indicated that the least hydrophobic peptide fractions interacted more strongly with choline on the liposome surfaces. The results suggested that the peptides were unequally distributed within the liposomes, even at the same encapsulation efficiency. These findings are important for designing delivery systems for commercial production of encapsulated peptides with improved functional attributes. PMID:27451165

  16. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions.

    Science.gov (United States)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco; Østergaard, Jesper

    2016-10-10

    Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations.

  17. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

    Energy Technology Data Exchange (ETDEWEB)

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  18. Antibody Peptide Based Antifungal Immunotherapy

    OpenAIRE

    Magliani, Walter; Conti, Stefania; Giovati, Laura; Zanello, Pier Paolo; Sperindè, Martina; Ciociola, Tecla; Polonelli, Luciano

    2012-01-01

    Fungal infections still represent relevant human illnesses worldwide and some are accompanied by unacceptably high mortality rates. The limited current availability of effective and safe antifungal agents makes the development of new drugs and approaches of antifungal vaccination/immunotherapy every day more needed. Among them, small antibody(Ab)-derived peptides are arousing great expectations as new potential antifungal agents. In this topic, the search path from the study of the yeast kill...

  19. Antimicrobial peptides in human sepsis

    Directory of Open Access Journals (Sweden)

    Lukas eMartin

    2015-08-01

    Full Text Available Nearly 100 years ago, antimicrobial peptides (AMPs were identified as an important part of innate immunity. They exist in species from bacteria to mammals and can be isolated in body fluids and on surfaces constitutively or induced by inflammation. Defensins have anti-bacterial effects against Gram-positive and Gram-negative bacteria as well as anti-viral and anti-yeast effects. Human neutrophil peptides (HNP 1-3 and human beta-defensins (HBDs 1-3 are some of the most important defensins in humans. Recent studies have demonstrated higher levels of HNP -1-3 and HBD-2 in sepsis. The bactericidal/permeability increasing protein (BPI attenuates local inflammatory response and decreases systemic toxicity of endotoxins. Moreover, BPI might reflect the severity of organ dysfunction in sepsis. Elevated plasma lactoferrin is detected in patients with organ failure. HNP-1-3, lactoferrin, BPI and heparin-binding protein (HBP are increased in sepsis. Human lactoferrin peptide 1-11 (hLF 1-11 possesses antimicrobial activity and modulates inflammation. The recombinant form of lactoferrin (talactoferrin alpha, TLF has been shown to decrease mortality in critically ill patients. A phase II/III study with TLF in sepsis did not confirm this result. The growing number of multiresistant bacteria is an ongoing problem in sepsis therapy. Furthermore, antibiotics are known to promote the liberation of pro-inflammatory cell components and thus augment the severity of sepsis. Compared to antibiotics, AMPs kill bacteria but also neutralize pathogenic factors such as lipopolysaccharide (LPS. The obstacle to applying naturally occurring AMPs is their high nephro- and neurotoxicity. Therefore, the challenge is to develop peptides to treat septic patients effectively without causing harm. This overview focuses on natural and synthetic AMPs in human and experimental sepsis and their potential to provide significant improvements in the treatment of critically ill with severe

  20. Antimicrobial Peptides: Versatile Biological Properties

    Directory of Open Access Journals (Sweden)

    Muthuirulan Pushpanathan

    2013-01-01

    Full Text Available Antimicrobial peptides are diverse group of biologically active molecules with multidimensional properties. In recent past, a wide variety of AMPs with diverse structures have been reported from different sources such as plants, animals, mammals, and microorganisms. The presence of unusual amino acids and structural motifs in AMPs confers unique structural properties to the peptide that attribute for their specific mode of action. The ability of these active AMPs to act as multifunctional effector molecules such as signalling molecule, immune modulators, mitogen, antitumor, and contraceptive agent makes it an interesting candidate to study every aspect of their structural and biological properties for prophylactic and therapeutic applications. In addition, easy cloning and recombinant expression of AMPs in heterologous plant host systems provided a pipeline for production of disease resistant transgenic plants. Besides these properties, AMPs were also used as drug delivery vectors to deliver cell impermeable drugs to cell interior. The present review focuses on the diversity and broad spectrum antimicrobial activity of AMPs along with its multidimensional properties that could be exploited for the application of these bioactive peptides as a potential and promising drug candidate in pharmaceutical industries.

  1. Antihypertensive Peptides from Milk Proteins

    Directory of Open Access Journals (Sweden)

    Heikki Vapaatalo

    2010-01-01

    Full Text Available Dietary proteins possess a wide range of nutritional and functional properties. They are used as a source of energy and amino acids, which are needed for growth and development. Many dietary proteins, especially milk proteins, contain physiologically active peptides encrypted in the protein sequence. These peptides may be released during gastrointestinal digestion or food processing and once liberated, cause different physiological functions. Milk-derived bioactive peptides are shown to have antihypertensive, antimicrobial, immunomodulatory, antioxidative and mineral-binding properties. During the fermentation of milk with certain lactobacilli, two interesting tripeptides Ile-Pro-Pro and Val-Pro-Pro are released from casein to the final product. These lactotripeptides have attenuated the development of hypertension in several animal models and lowered blood pressure in clinical studies. They inhibit ACE in vitro at micromolar concentrations, protect endothelial function in vitro and reduce arterial stiffness in humans. Thus, milk as a traditional food product can after certain processing serve as a functional food and carry specific health-promoting effects, providing an option to control blood pressure.

  2. Chemical Methods for Peptide and Protein Production

    Directory of Open Access Journals (Sweden)

    Istvan Toth

    2013-04-01

    Full Text Available Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported a-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

  3. Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lisa Gerner

    2016-09-01

    Full Text Available Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2 has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501 in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1].

  4. Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis

    DEFF Research Database (Denmark)

    Maga, G; Mossi, R; Fischer, R;

    1997-01-01

    that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF......-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the formation...

  5. Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: calmodulin dendrograms show significant lack of parallelism

    Science.gov (United States)

    Nakayama, S.; Kretsinger, R. H.

    1993-01-01

    In the first report in this series we presented dendrograms based on 152 individual proteins of the EF-hand family. In the second we used sequences from 228 proteins, containing 835 domains, and showed that eight of the 29 subfamilies are congruent and that the EF-hand domains of the remaining 21 subfamilies have diverse evolutionary histories. In this study we have computed dendrograms within and among the EF-hand subfamilies using the encoding DNA sequences. In most instances the dendrograms based on protein and on DNA sequences are very similar. Significant differences between protein and DNA trees for calmodulin remain unexplained. In our fourth report we evaluate the sequences and the distribution of introns within the EF-hand family and conclude that exon shuffling did not play a significant role in its evolution.

  6. New insight into molecular phylogeny and epidemiology of Sporothrix schenckii species complex based on calmodulin-encoding gene analysis of Italian isolates.

    Science.gov (United States)

    Romeo, Orazio; Scordino, Fabio; Criseo, Giuseppe

    2011-09-01

    In this study, we investigated phylogenetic relationships among Italian Sporothrix schenckii isolates, by comparing their partial calmodulin sequences. In this analysis, we used 26 environmental strains of S. schenckii, plus two autochthonous clinical isolates. The results showed that our clinical strains grouped with S. schenckii sensu stricto isolates, whereas all 26 environmental isolates co-clustered with Sporothrix albicans (now regarded as a synonym of Sporothrix pallida), a non-pathogenic species closely related to S. schenckii. Furthermore, the group of environmental strains was found to be quite heterogeneous and further subdivided into two subgroups. The data reported here also showed that molecular methods, for specific identification of S. schenckii, developed before the description of its closely related species should be used with caution because of the possibility of false positive results, which could lead to inappropriate antifungal therapy. This study improves our understanding of the distribution of these new closely related Sporothrix species which also showed significant differences in antifungal susceptibilities.

  7. MICrocephaly, disproportionate pontine and cerebellar hypoplasia syndrome: A clinico-radiologic phenotype linked to calcium/calmodulin-dependent serine protein kinase gene mutation

    Directory of Open Access Journals (Sweden)

    Rashid Saleem

    2013-01-01

    Full Text Available MICrocephaly, disproportionate pontine and cerebellar hypoplasia (MICPCH syndrome, a rare X-linked disorder, generally seen in girls, is characterized by neurodevelopmental delay, microcephaly, and disproportionate pontine and cerebellar hypoplasia. It is caused by inactivating calcium/calmodulin-dependent serine protein kinase (CASK gene mutations. We report a 2-year-old girl with severe neurodevelopmental delay, microcephaly, minimal pontine hypoplasia, cerebellar hypoplasia, and normal looking corpus callosum, with whom the conventional cytogenetic studies turned out to be normal, and an array-comparative genomic hybridization (a-CGH analysis showed CASK gene duplication at Xp11.4. Our case highlights the importance of using clinico-radiologic phenotype to guide genetic investigation and it also confirms the role of a-CGH analysis in establishing the genetic diagnosis of MICPCH syndrome, when conventional cytogenetic studies are inconclusive.

  8. Human Antimicrobial Peptides and Proteins

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2014-05-01

    Full Text Available As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32 can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized

  9. Molecular analysis of the graviperception signal transduction in the flagellate Euglena gracilis: Involvement of a transient receptor potential-like channel and a calmodulin

    Science.gov (United States)

    Häder, Donat-Peter; Richter, Peter R.; Schuster, Martin; Daiker, Viktor; Lebert, Michael

    2009-04-01

    Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavior responses. The organism shows pronounced negative gravitaxis. This movement is based on physiological mechanisms, which in the past had been only indirectly assessed. It was shown that mechano-sensitive calcium channels are involved in the gravitaxis response. Recent studies have demonstrated that members of the transient receptor potential (TRP) family function as mechano-sensitive channels in several different cell types. We have sequenced part of a TRP gene in Euglena and applied RNA interference (RNAi) to confirm that these channels are involved in graviperception. It was found that RNAi against the putative TRP channel abolished gravitaxis. The genes of three calmodulins were sequences in Euglena, one of which was previously known in its protein structure (cal 1). The other two were unknown (cal 2 and cal 3). Cal 2 has been analyzed in detail. The biosynthesis of the corresponding proteins of cal 1 and cal 2 was inhibited by means of RNA interference to see whether this blockage impairs gravitaxis. RNAi of cal 1 leads to a long-term loss of free swimming in the cells (while euglenoid movement persists). It induced pronounced cell form aberrations and the division of cells was hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Thus cal 1 does not seem to be involved in gravitaxis. In contrast, the blockage of cal 2 has no pronounced influence on motility and cell form but leads to a complete loss of gravitactic orientation for more than 30 days showing that this calmodulin is an element in the signal transduction chain. The data are discussed in the context of the current model of the gravitaxis signal transduction chain in Euglena gracilis.

  10. Phylogeny of plant calcium and calmodulin-dependent protein kinases (CCaMKs and functional analyses of tomato CCaMK in disease resistance

    Directory of Open Access Journals (Sweden)

    Ji-Peng eWang

    2015-12-01

    Full Text Available Calcium and calmodulin-dependent protein kinase (CCaMK is a member of calcium/calmodulin-dependent protein kinase superfamily and is essential to microbe- plant symbiosis. To date, the distribution of CCaMK gene in plants has not yet been completely understood, and its function in plant disease resistance remains unclear. In this study, we systemically identified the CCaMK genes in genomes of 44 plant species in Phytozome and analyzed the function of tomato CCaMK (SlCCaMK in resistance to various pathogens. CCaMKs in 18 additional plant species were identified, yet the absence of CCaMK gene in green algae and cruciferous species was confirmed. Sequence analysis of full-length CCaMK proteins from 44 plant species demonstrated that plant CCaMKs are highly conserved across all domains. Most of the important regulatory amino acids are conserved throughout all sequences, with the only notable exception being observed in N-terminal autophosphorylation site corresponding to Ser 9 in the Medicago truncatula CCaMK. CCaMK gene structures are similar, mostly containing six introns with a phase profile of 200200 and the exception was only noticed at the first exons. Phylogenetic analysis demonstrated that CCaMK lineage is likely to have diverged early from a calcium-dependent protein kinase (CDPK gene in the ancestor of all nonvascular plant species. The SlCCaMK gene was widely and differently responsive to diverse pathogenic stimuli. Furthermore, knock-down of SlCCaMK reduced tomato resistance to Sclerotinia sclerotiorum and Pseudomonas syringae pv. tomato (Pst DC3000 and decreased H2O2 accumulation in response to Pst DC3000 inoculation. Our results reveal that SlCCaMK positively regulates disease resistance in tomato via promoting H2O2 accumulation. SlCCaMK is the first CCaMK gene proved to function in plant disease resistance.

  11. Molecular imaging probes derived from natural peptides.

    Science.gov (United States)

    Charron, C L; Hickey, J L; Nsiama, T K; Cruickshank, D R; Turnbull, W L; Luyt, L G

    2016-06-01

    Covering: up to the end of 2015.Peptides are naturally occurring compounds that play an important role in all living systems and are responsible for a range of essential functions. Peptide receptors have been implicated in disease states such as oncology, metabolic disorders and cardiovascular disease. Therefore, natural peptides have been exploited as diagnostic and therapeutic agents due to the unique target specificity for their endogenous receptors. This review discusses a variety of natural peptides highlighting their discovery, endogenous receptors, as well as their derivatization to create molecular imaging agents, with an emphasis on the design of radiolabelled peptides. This review also highlights methods for discovering new and novel peptides when knowledge of specific targets and endogenous ligands are not available. PMID:26911790

  12. Use of Galerina marginata genes and proteins for peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  13. Antimicrobial Peptides in Toroidal and Cylindrical Pores

    OpenAIRE

    Mihajlovic, Maja; Lazaridis, Themis

    2010-01-01

    Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or ...

  14. Interaction of small peptides with lipid bilayers.

    OpenAIRE

    Damodaran, K. V.; Merz, K M; Gaber, B P

    1995-01-01

    Molecular dynamics simulations of the tripeptide Ala-Phe-Ala-O-tert-butyl interacting with dimyristoylphosphatidylcholine lipid bilayers have been carried out. The lipid and aqueous environments of the peptide, the alkyl chain order, and the lipid and peptide dynamics have been investigated with use of density profiles, radial distribution functions, alkyl chain order parameter profiles, and time correlation functions. It appears that the alkyl chain region accommodates the peptides in the bi...

  15. Self-assembly of tetraphenylalanine peptides

    OpenAIRE

    Mayans Tayadella, Enric; Ballano Ballano, María Gema; Casanovas Salas, Jordi; Díaz Andrade, Angélica María; Pérez Madrigal, Maria del Mar; Estrany Coda, Francesc; Puiggalí Bellalta, Jordi; Cativiela Marín, Carlos A.; Alemán Llansó, Carlos

    2015-01-01

    Three different tetraphenylalanine (FFFF) based peptides that differ at the N- and C-termini have been synthesized by using standard procedures to study their ability to form different nanoassemblies under a variety of conditions. The FFFF peptide assembles into nanotubes that show more structural imperfections at the surface than those formed by the diphenylalanine (FF) peptide under the same conditions. Periodic DFT calculations (M06L functional) were used to propose a model that consists o...

  16. Salt-resistant short antimicrobial peptides.

    Science.gov (United States)

    Mohanram, Harini; Bhattacharjya, Surajit

    2016-05-01

    Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactivation. The high cost of chemical synthesis of AMPs also impedes potential clinical application. Consequently, short AMPs resistant toward salt and serum inactivation are desirable for the development of peptide antibiotics. In this work, we designed a 12-residue amphipathic helical peptide RR12 (R-R-L-I-R-L-I-L-R-L-L-R-amide) and two Trp containing analogs of RR12 namely RR12Wpolar (R-R-L-I-W-L-I-L-R-L-L-R-amide), and RR12Whydro (R-R-L-I-R-L-W-L-R-L-L-R-amide). Designed peptides demonstrated potent antibacterial activity; MIC ranging from 2 to 8 μM, in the presence of sodium chloride (150 mM and 300 mM). Antibacterial activity of these peptides was also detected in the presence of human serum. Designed peptides, in particular RR12 and RR12Whydro, were only poorly hemolytic. As a mode of action; these peptides demonstrated efficient permeabilization of bacterial cell membrane and lysis of cell structure. We further investigated interactions of the designed peptides with lipopolysaccharide (LPS), the major component of the outer membrane permeability barrier of Gram-negative bacteria. Designed peptides adopted helical conformations in complex with LPS. Binding of peptides with LPS has yielded dissociation the aggregated structures of LPS. Collectively, these designed peptides hold ability to be developed for salt-resistant antimicrobial compounds. Most importantly, current work provides insights for designing salt-resistant antimicrobial peptides. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 345-356, 2016. PMID:26849911

  17. Genome-based peptide fingerprint scanning

    OpenAIRE

    Giddings, Michael C.; Shah, Atul A.; Gesteland, Ray; Moore, Barry

    2002-01-01

    We have implemented a method that identifies the genomic origins of sample proteins by scanning their peptide-mass fingerprint against the theoretical translation and proteolytic digest of an entire genome. Unlike previously reported techniques, this method requires no predefined ORF or protein annotations. Fixed-size windows along the genome sequence are scored by an equation accounting for the number of matching peptides, the number of missed enzymatic cleavages in each peptide, the number ...

  18. The Function and Development of Soybean Peptides

    Institute of Scientific and Technical Information of China (English)

    Yang Caiyan; Song Junmei

    2009-01-01

    Soybean peptides are small molecules hydrolyzed soy protein,from three to six amino acid composition of the peptide mixture,in 1000Da molecular weight below.Because it has a lot of good physical and chemical properties and physiological functions,in many areas has been widely used.This paper reviews the soybean peptide physical and chemical characteristics,physiological functions,technology and applications in the food industry.

  19. Insect inducible antimicrobial peptides and their applications.

    Science.gov (United States)

    Ezzati-Tabrizi, Reyhaneh; Farrokhi, Naser; Talaei-Hassanloui, Reza; Alavi, Seyed Mehdi; Hosseininaveh, Vahid

    2013-12-01

    Antimicrobial peptides (AMPs) are found as important components of the innate immune system (host defense) of all invertebrates. These peptides can be constitutively expressed or induced in response to microbial infections. Indeed, they vary in their amino acid sequences, potency and antimicrobial activity spectra. The smaller AMPs act greatly by disrupting the structure or function of microbial cell membranes. Here, the insect innate immune system with emphasis on inducible antimicrobial peptide properties against microbial invaders has been discussed.

  20. Modulation of autoimmunity with artificial peptides

    Science.gov (United States)

    La Cava, Antonio

    2010-01-01

    The loss of immune tolerance to self antigens leads to the development of autoimmune responses. Since self antigens are often multiple and/or their sequences may not be known, one approach to restore immune tolerance uses synthetic artificial peptides that interfere or compete with self peptides in the networks of cellular interactions that drive the autoimmune process. This review describes the rationale behind the use of artificial peptides in autoimmunity and their mechanisms of action. Examples of use of artificial peptides in preclinical studies and in the management of human autoimmune diseases are provided. PMID:20807590

  1. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending...... of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden....

  2. Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity

    DEFF Research Database (Denmark)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Tesser, G.I.;

    1997-01-01

    or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin...

  3. Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Langham, Allison A; Kaznessis, Yiannis N

    2006-01-01

    peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural...

  4. Peptide Nucleic Acids Complexes of Two Peptide Nucleic Acid Strands and One

    DEFF Research Database (Denmark)

    1999-01-01

    Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest...

  5. Mechanism and kinetics of peptide partitioning into membranes from all-atom simulations of thermostable peptides

    OpenAIRE

    Ulmschneider, Martin B.; Doux, Jacques P F; Killian, J. Antoinette; Smith, Jeremy C.; Ulmschneider, Jakob P.

    2010-01-01

    Partitioning properties of transmembrane (TM) polypeptide segments directly determine membrane protein folding, stability, and function, and their understanding is vital for rational design of membrane active peptides. However, direct determination of water-to-bilayer transfer of TM peptides has proved difficult. Experimentally, sufficiently hydrophobic peptides tend to aggregate, while atomistic computer simulations at physiological temperatures cannot yet reach the long time scales required...

  6. Glucagon-like peptide-1

    DEFF Research Database (Denmark)

    Deacon, C F; Holst, Jens Juul; Carr, R D

    1999-01-01

    Type 2 diabetes mellitus is a metabolic disease resulting in raised blood sugar which, if not satisfactorily controlled, can cause severe and often debilitating complications. Unfortunately, for many patients, the existing therapies do not give adequate control. Glucagon-like peptide-1 (GLP-1) is...... an incretin hormone which has a spectrum of activities which oppose the symptoms of diabetes. Of particular significance is the fact that these actions are glucose-dependent, meaning that the risk of severe hypoglycemia is practically eliminated. The recent elucidation of the key role of dipeptidyl...

  7. Atrial natriuretic peptides in plasma

    DEFF Research Database (Denmark)

    Goetze, Jens P; Holst Hansen, Lasse; Terzic, Dijana;

    2015-01-01

    Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone....... These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid...

  8. Dendritic spine changes in the development of alcohol addiction regulated by α-calcium/calmodulin-dependent protein kinase II

    Directory of Open Access Journals (Sweden)

    Zofia Mijakowska

    2014-03-01

    Full Text Available Introduction Alcohol has many adverse effects on the brain. Among them are dendritic spine morphology alterations, which are believed to be the basis of alcohol addiction. Autophosphorylation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII has been shown to regulate spine morphology in vitro. Here we show that αCaMKII can also regulate addiction related behaviour and dendritic spine morphology changes caused by alcohol consumption in vivo. Method 12 αCaMKII-autophosphorylation deficient female mice (T286A and 12 wild type littermates were used in the study. T286A strain was created by Giese et al. (1998. Mice were housed and tested in two IntelliCages from NewBehavior (www.newbehavior.com. IntelliCage is an automated learning system. After 95 days of alcohol drinking interrupted by tests for motivation, persistence in alcohol seeking and probability of relapse, mice were ascribed to ‘high’ or ‘low’ drinkers group according to their performance in the tests. Additional criterion was the amount of alcohol consumed during the whole experiment. Result of each test was evaluated separately. 1/3 of the mice that scored highest in each criterion were considered ‘positive’ for this trait. ‘Positive’ animals were given 1 point, negative 0 points. Mice that were positive in at least 2 criteria were ascribed to ‘high’ drinkers (‘+’ group. Remaining mice – to ‘low’ drinkers (‘–‘. This method of behavioral phenotyping, developed by Radwanska and Kaczmarek (2012, is inspired by DSM-IV. Since the results of this evaluation are discrete (i.e. by definition all the animals score between 0 to +4, we developed also a continuous method of addiction rating, which we call ‘addiction index’. The result of the second method is a sum of the standardized (z-score results of the above mentioned tests. We use it to examine the correlations between addiction-like behavior and spine parameters. Control group (12 WT, 8

  9. Antimicrobial peptides in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    A Bogaerts

    2010-01-01

    Full Text Available The nematode Caenorhabditis elegans is one of the most successful model species for experimental research because of its sequenced genome, the versatile genetic toolkit and the straightforward breeding among others. In natural conditions however, this tiny worm is constantly surrounded by micro-organisms, simultaneously a source of indispensable nutrition and inevitable pathogens. Lacking an adaptive immune system, the worm solely relies on its innate immune defence to cope with its challenging life style. Hence C. elegans is an excellent model to gain more insight in innate immunity, which is remarkably preserved between invertebrate and vertebrate animals. The innate defence consists of receptors to detect potential pathogens, a complex network of signalling pathways and last but not least, effector molecules to abolish harmful microbes. In this review, we focus on the antimicrobial peptides, a vital subgroup of effector molecules. We summarise the current knowledge of the different families of C. elegans antimicrobial peptides, comprising NLPs, caenacins, ABFs, caenopores, and a recently discovered group with antifungal activity among which thaumatin-like proteins.

  10. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans;

    1990-01-01

    not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...... for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we...

  11. Determination of peptide content of DOTA-peptides by metal titration and UPLC

    International Nuclear Information System (INIS)

    Radiolabelled DOTA-peptides are in use for Peptide Receptor Radionuclide Scintigraphy (PRS) and Therapy (PRRT), e.g with 177Lu-DOTA-TATE or 90Y-DOTATOC. Labelling conditions are frequently critical. Therefore, the ingredients of the reaction, e.g. radiometal (90Y and 177Lu) and DOTA-peptide should be pure and the content known. Quality control of DOTA-peptide, can be performed with various methods, most commonly by UV. There are numerous conditions in which this is hampered, e.g. impurities may also have UV-absorption. The aim of the study was to quantify content and purity of DOTA-peptide

  12. Engineered Adhesion Peptides for Improved Silicon Adsorption.

    Science.gov (United States)

    Ramakrishnan, Sathish Kumar; Jebors, Said; Martin, Marta; Cloitre, Thierry; Agarwal, Vivechana; Mehdi, Ahmad; Martinez, Jean; Subra, Gilles; Gergely, Csilla

    2015-11-01

    Engineering peptides that present selective recognition and high affinity for a material is a major challenge for assembly-driven elaboration of complex systems with wide applications in the field of biomaterials, hard-tissue regeneration, and functional materials for therapeutics. Peptide-material interactions are of vital importance in natural processes but less exploited for the design of novel systems for practical applications because of our poor understanding of mechanisms underlying these interactions. Here, we present an approach based on the synthesis of several truncated peptides issued from a silicon-specific peptide recovered via phage display technology. We use the photonic response provided by porous silicon microcavities to evaluate the binding efficiency of 14 different peptide derivatives. We identify and engineer a short peptide sequence (SLVSHMQT), revealing the highest affinity for p(+)-Si. The molecular recognition behavior of the obtained peptide fragment can be revealed through mutations allowing identification of the preferential affinity of certain amino acids toward silicon. These results constitute an advance in both the engineering of peptides that reveal recognition properties for silicon and the understanding of biomolecule-material interactions.

  13. [Application on food preservative of antimicrobial peptides].

    Science.gov (United States)

    Zhao, Hongyan; Mu, Yu; Zhao, Baohua

    2009-07-01

    Antimicrobial peptides are an integral component of the innate immune system, it can counteract outer membrane pathogen such as bacteria, fungi, viruses, protozoan and so on. Owing to the sterilization and innocuity, it has the potential to be crude food preservative. In this paper the uses of antibacterial peptides in the food preservative were analyzed.

  14. Prediction of twin-arginine signal peptides

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Nielsen, Henrik; Widdick, D.;

    2005-01-01

    peptides and 84% of the annotated cleavage sites of these Tat signal peptides were correctly predicted. This method generates far less false positive predictions on various datasets than using simple pattern matching. Moreover, on the same datasets TatP generates less false positive predictions than...

  15. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are...

  16. Peptide Mass Fingerprinting of Egg White Proteins

    Science.gov (United States)

    Alty, Lisa T.; LaRiviere, Frederick J.

    2016-01-01

    Use of advanced mass spectrometry techniques in the undergraduate setting has burgeoned in the past decade. However, relatively few undergraduate experiments examine the proteomics tools of protein digestion, peptide accurate mass determination, and database searching, also known as peptide mass fingerprinting. In this experiment, biochemistry…

  17. New Biodegradable Peptide-based Polymer Constructs

    NARCIS (Netherlands)

    van Dijk, M.

    2009-01-01

    Peptide-based polymers are of increasing interest, since they can be applied for a variety of purposes such as drug delivery devices, scaffolds for tissue engineering and -repair, and as novel biomaterials. Peptide-based polymers are common in nature and often exhibit special characteristics. Howeve

  18. Protein identification by peptide mass fingerprinting

    DEFF Research Database (Denmark)

    Hjernø, Karin

    2007-01-01

      Peptide mass fingerprinting is an effective way of identifying, e.g., gel-separated proteins, by matching experimentally obtained peptide mass data against large databases. However, several factors are known to influence the quality of the resulting matches, such as proteins contaminating the s...

  19. Peptidomic Identification of Serum Peptides Diagnosing Preeclampsia.

    Directory of Open Access Journals (Sweden)

    Qiaojun Wen

    Full Text Available We sought to identify serological markers capable of diagnosing preeclampsia (PE. We performed serum peptide analysis (liquid chromatography mass spectrometry of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100% and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%. The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA, 1 from alpha-1-antitrypsin (A1AT, 1 from apolipoprotein L1 (APO-L1, 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4, 2 from kininogen-1 (KNG1, and 1 from thymosin beta-4 (TMSB4. We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.

  20. B-Type allatostatins and sex peptides

    Science.gov (United States)

    In many species, mating induces a number of behavioral changes in the female. For Drosophila melanogaster, the sex peptide (SP) has been identified as the main molecular factor behind these responses. Recently, the sex peptide receptor (SPR), a GPCR activated by SP has also been characterized as res...

  1. Trandermal Peptides for Large Molecule Delivery

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ A research team, led by Prof. WEN Longping from the University of Science and Technology of China under CAS,has successfully screened out a trandermal peptide, using biotechnology. The new peptide is able to deliver insulin into human body through skin, rendering an immediate therapeutic effect. The finding was published in the March 27 issue of the journal Natural Biotechnology.

  2. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...

  3. Effect of Calmodulin on the Differrentiation and Migration of PC12 Cells%钙调蛋白在PC12细胞分化和迁移中的作用

    Institute of Scientific and Technical Information of China (English)

    袁俊; 李朝军

    2011-01-01

    To investigate the roles of calmodulin during neuronal differentiation and migration,we checked PC12 cells by immunofluorescence staining and single cell tracking assay after NGF treatment. We found that calmodulin showed a dense distribution pattern in top of PC12 cells. Only a small percentage of the cells grown in W7 treatment cells. A single cell tracking experiment showed that calmodulin in PC12 cells could increase cell motility. The data suggested that calmodulin may play an important role in differentiation and migration of PC12 cells.%PC12 细胞经神经生长因子 (NGF)作用后,利用免疫荧光染色、单个细胞迁移率检测等方法,研究了钙调蛋白在PC12 细胞中的分布以及钙调蛋白对PC12 细胞分化和迁移的影响.免疫荧光染色结果表明,钙调蛋白在PC12细胞突起的顶端处呈密集分布.加入钙调蛋白抑制剂W7的细胞仅有少量长出突起.单个细胞迁移率检测表明钙调蛋白可能促进PC12 细胞迁移.提示钙调蛋白可能在PC12细胞的分化和迁移过程中发挥作用.

  4. Antioxidant activity of yoghurt peptides: Part 2 – Characterisationof peptide fractions

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Baron, Caroline; Nielsen, Nina Skall;

    2010-01-01

    The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid...... antioxidant activity in these fractions.......The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid...... the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained...

  5. Role of peptide bond in the realization of biological activity of short peptides.

    Science.gov (United States)

    Khavinson, V Kh; Tarnovskaya, S I; Lin'kova, N S; Chervyakova, N A; Nichik, T E; Elashkina, E V; Chalisova, N I

    2015-02-01

    We performed a comparative analysis of biological activity of Lys-Glu peptide and its amino acid constituents. It was established that Lys-Glu stimulated proliferation of splenic cells in organotypic culture, while the mixture of glutamic acid and lysine inhibited culture growth. Using the method of molecular docking, we showed that glutamic acid, lysine, and Lys-Glu peptide can interact with different DNA sequences. The energy of interaction and the most beneficial localization of glutamic acid, lysine, and Lys-Glu peptide in DNA molecule was calculated. We demonstrated the interaction of the peptide and amino acids with DNA along the minor groove. The energy of DNA interaction with the peptide is higher than with individual amino acids. The peptide bonds increase the interaction of Lys-Glu peptide with DNA, which potentiates the biological effect on cell proliferation in organotypic culture of splenic cells.

  6. Peptide nanospheres self-assembled from a modified β-annulus peptide of Sesbania mosaic virus.

    Science.gov (United States)

    Matsuura, Kazunori; Mizuguchi, Yusaku; Kimizuka, Nobuo

    2016-11-01

    A novel β-annulus peptide of Sesbania mosaic virus bearing an FKFE sequence at the C terminus was synthesized, and its self-assembling behavior in water was investigated. Dynamic light scattering and transmission electron microscopy showed that the β-annulus peptide bearing an FKFE sequence self-assembled into approximately 30 nm nanospheres in water at pH 3.8, whereas the β-annulus peptide without the FKFE sequence afforded only irregular aggregates. The peptide nanospheres possessed a definite critical aggregation concentration (CAC = 26 μM), above which the size of nanospheres were nearly unaffected by the peptide concentration. The formation of peptide nanospheres was significantly affected by pH; the peptide did not form any assemblies at pH 2.2, whereas larger aggregates were formed at pH 6.4-11.6. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 470-475, 2016. PMID:26573103

  7. Design of Asymmetric Peptide Bilayer Membranes.

    Science.gov (United States)

    Li, Sha; Mehta, Anil K; Sidorov, Anton N; Orlando, Thomas M; Jiang, Zhigang; Anthony, Neil R; Lynn, David G

    2016-03-16

    Energetic insights emerging from the structural characterization of peptide cross-β assemblies have enabled the design and construction of robust asymmetric bilayer peptide membranes. Two peptides differing only in their N-terminal residue, phosphotyrosine vs lysine, coassemble as stacks of antiparallel β-sheets with precisely patterned charged lattices stabilizing the bilayer leaflet interface. Either homogeneous or mixed leaflet composition is possible, and both create nanotubes with dense negative external and positive internal solvent exposed surfaces. Cross-seeding peptide solutions with a preassembled peptide nanotube seed leads to domains of different leaflet architecture within single nanotubes. Architectural control over these cross-β assemblies, both across the bilayer membrane and along the nanotube length, provides access to highly ordered asymmetric membranes for the further construction of functional mesoscale assemblies.

  8. Intracellular signalling by C-peptide.

    Science.gov (United States)

    Hills, Claire E; Brunskill, Nigel J

    2008-01-01

    C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na(+)/K(+) ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes. PMID:18382618

  9. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  10. Antimicrobial peptides important in innate immunity.

    Science.gov (United States)

    Cederlund, Andreas; Gudmundsson, Gudmundur H; Agerberth, Birgitta

    2011-10-01

    Antimicrobial peptides are present in all walks of life, from plants to animals, and they are considered to be endogenous antibiotics. In general, antimicrobial peptides are determinants of the composition of the microbiota and they function to fend off microbes and prevent infections. Antimicrobial peptides eliminate micro-organisms through disruption of their cell membranes. Their importance in human immunity, and in health as well as disease, has only recently been appreciated. The present review provides an introduction to the field of antimicrobial peptides in general and discusses two of the major classes of mammalian antimicrobial peptides: the defensins and the cathelicidins. The review focuses on their structures, their main modes of action and their regulation.

  11. Modelling water molecules inside cyclic peptide nanotubes

    Science.gov (United States)

    Tiangtrong, Prangsai; Thamwattana, Ngamta; Baowan, Duangkamon

    2016-03-01

    Cyclic peptide nanotubes occur during the self-assembly process of cyclic peptides. Due to the ease of synthesis and ability to control the properties of outer surface and inner diameter by manipulating the functional side chains and the number of amino acids, cyclic peptide nanotubes have attracted much interest from many research areas. A potential application of peptide nanotubes is their use as artificial transmembrane channels for transporting ions, biomolecules and waters into cells. Here, we use the Lennard-Jones potential and a continuum approach to study the interaction of a water molecule in a cyclo[(- D-Ala- L-Ala)_4-] peptide nanotube. Assuming that each unit of a nanotube comprises an inner and an outer tube and that a water molecule is made up of a sphere of two hydrogen atoms uniformly distributed over its surface and a single oxygen atom at the centre, we determine analytically the interaction energy of the water molecule and the peptide nanotube. Using this energy, we find that, independent of the number of peptide units, the water molecule will be accepted inside the nanotube. Once inside the nanotube, we show that a water molecule prefers to be off-axis, closer to the surface of the inner nanotube. Furthermore, our study of two water molecules inside the peptide nanotube supports the finding that water molecules form an array of a 1-2-1-2 file inside peptide nanotubes. The theoretical study presented here can facilitate thorough understanding of the behaviour of water molecules inside peptide nanotubes for applications, such as artificial transmembrane channels.

  12. Encapsulation of Enzymes and Peptides

    Science.gov (United States)

    Meesters, Gabrie M. H.

    A large part of formulated peptides and proteins, e.g., enzymes used as food ingredients, are formulated in a liquid form. Often, they are dissolved in water to which glycerol or sorbitol is added to reduce the water activity of the liquid, thus reducing the change of microbial growth. Still, there are reasons to formulate them in a solid form. Often, these reasons are stability, since a dry formulation is often much better than liquid formulations, and less transportation cost, since less mass is transported if one gets rid of the liquid; however, most of the times, the reason is that the product is mixed with a solid powder. Here, a liquid addition would lead to lump formation.

  13. Antimicrobial peptides of multicellular organisms

    Science.gov (United States)

    Zasloff, Michael

    2002-01-01

    Multicellular organisms live, by and large, harmoniously with microbes. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes or antibodies. A plant seed germinates successfully in the midst of soil microbes. How is this accomplished? Both animals and plants possess potent, broad-spectrum antimicrobial peptides, which they use to fend off a wide range of microbes, including bacteria, fungi, viruses and protozoa. What sorts of molecules are they? How are they employed by animals in their defence? As our need for new antibiotics becomes more pressing, could we design anti-infective drugs based on the design principles these molecules teach us?

  14. Antimicrobial peptides in the brain.

    Science.gov (United States)

    Su, Yanhua; Zhang, Kai; Schluesener, Hermann J

    2010-10-01

    Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune system of many species. The brain is an immunologically privileged organ but can produce a robust immune response against pathogens and cell debris, promoting rapid and efficient clearance. AMPs may be critically involved in the innate immune system of the brain. Though the mechanisms of AMPs' action in the brain still need further elucidation, many studies have shown that AMPs are multifunctional molecules in the brain. In addition to antimicrobial action, they take part in congenital and adaptive immune reactions (immunoregulation), function as signaling molecules in tissue repair, inflammation and other important processes through different mechanisms, and they might, in addition, become diagnostic markers of brain disease.

  15. The novel amyloid-beta peptide aptamer inhibits intracellular amyloid-beta peptide toxicity

    Institute of Scientific and Technical Information of China (English)

    Xu Wang; Yi Yang; Mingyue Jia; Chi Ma; Mingyu Wang; Lihe Che; Yu Yang; Jiang Wu

    2013-01-01

    Amyloid β peptide binding alcohol dehydrogenase (ABAD) decoy peptide (DP) can competitively antagonize binding of amyloid β peptide to ABAD and inhibit the cytotoxic effects of amyloid β peptide. Based on peptide aptamers, the present study inserted ABAD-DP into the disulfide bond of human thioredoxin (TRX) using molecular cloning technique to construct a fusion gene that can express the TRX1-ABAD-DP-TRX2 aptamer. Moreover, adeno-associated virus was used to allow its stable expression. Immunofluorescent staining revealed the co-expression of the transduced fusion gene TRX1-ABAD-DP-TRX2 and amyloid β peptide in NIH-3T3 cells, indicating that the TRX1-ABAD-DP-TRX2 aptamer can bind amyloid β peptide within cells. In addition, cell morphology and MTT results suggested that TRX1-ABAD-DP-TRX2 attenuated amyloid β peptide-induced SH-SY5Y cell injury and improved cell viability. These findings confirmed the possibility of constructing TRX-based peptide aptamer using ABAD-DP. Moreover, TRX1-ABAD-DP-TRX2 inhibited the cytotoxic effect of amyloid β peptide.

  16. Interactions of Bio-Inspired Membranes with Peptides and Peptide-Mimetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Michael Sebastiano

    2015-08-01

    Full Text Available Via Dissipative Particle Dynamics (DPD and implicit solvent coarse-grained (CG Molecular Dynamics (MD we examine the interaction of an amphiphilic cell-penetrating peptide PMLKE and its synthetic counterpart with a bio-inspired membrane. We use the DPD technique to investigate the interaction of peptide-mimetic nanoparticles, or nanopins, with a three-component membrane. The CG MD approach is used to investigate the interaction of a cell-penetrating peptide PMLKE with single-component membrane. We observe the spontaneous binding and subsequent insertion of peptide and nanopin in the membrane by using CG MD and DPD approaches, respectively. In addition, we find that the insertion of peptide and nanopins is mainly driven by the favorable enthalpic interactions between the hydrophobic components of the peptide, or nanopin, and the membrane. Our study provides insights into the mechanism underlying the interactions of amphiphilic peptide and peptide-mimetic nanoparticles with a membrane. The result of this study can be used to guide the functional integration of peptide and peptide-mimetic nanoparticles with a cell membrane.

  17. Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

    Vegt, Erik; Eek, Annemarie; Oyen, Wim J.G.; Gotthardt, Martin; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine (444), PO Box 9101, Nijmegen (Netherlands); Jong, Marion de [Erasmus Medical Centre, Department of Nuclear Medicine, Rotterdam (Netherlands)

    2010-02-15

    In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin. Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of {sup 111}In-albumin, {sup 111}In-minigastrin, {sup 111}In-exendin and {sup 111}In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of {sup 111}In-minigastrin, {sup 111}In-exendin and {sup 111}In-octreotide was determined. FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of {sup 111}In-albumin, {sup 111}In-exendin and {sup 111}In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide 6), was selected for in vivo testing. In rats, 5 mg of peptide 6 very efficiently inhibited the renal uptake of {sup 111}In-minigastrin, by 88%. Uptake of {sup 111}In-exendin and {sup 111}In-octreotide was reduced by 26 and 33%, respectively. The albumin-derived peptide 6 efficiently inhibited the renal reabsorption of {sup 111}In-minigastrin, {sup 111}In-exendin and {sup 111}In-octreotide and is a promising candidate for kidney protection in PRRT. (orig.)

  18. rapmad: Robust analysis of peptide microarray data

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    Rothermel Andrée

    2011-08-01

    Full Text Available Abstract Background Peptide microarrays offer an enormous potential as a screening tool for peptidomics experiments and have recently seen an increased field of application ranging from immunological studies to systems biology. By allowing the parallel analysis of thousands of peptides in a single run they are suitable for high-throughput settings. Since data characteristics of peptide microarrays differ from DNA oligonucleotide microarrays, computational methods need to be tailored to these specifications to allow a robust and automated data analysis. While follow-up experiments can ensure the specificity of results, sensitivity cannot be recovered in later steps. Providing sensitivity is thus a primary goal of data analysis procedures. To this end we created rapmad (Robust Alignment of Peptide MicroArray Data, a novel computational tool implemented in R. Results We evaluated rapmad in antibody reactivity experiments for several thousand peptide spots and compared it to two existing algorithms for the analysis of peptide microarrays. rapmad displays competitive and superior behavior to existing software solutions. Particularly, it shows substantially improved sensitivity for low intensity settings without sacrificing specificity. It thereby contributes to increasing the effectiveness of high throughput screening experiments. Conclusions rapmad allows the robust and sensitive, automated analysis of high-throughput peptide array data. The rapmad R-package as well as the data sets are available from http://www.tron-mz.de/compmed.

  19. Conus Peptides A Rich Pharmaceutical Treasure

    Institute of Scientific and Technical Information of China (English)

    Cheng-Zhong WANG; Cheng-Wu CHI

    2004-01-01

    Marine predatory cone snails (genus Conus) with over 500 species represent what is arguably the largest single genus of marine animals alive today. All Conus are venomous and utilize a complex mixture of Conus peptides to capture their preys and for other biological purposes. Each component of Conus peptides selectively targets a specific subtype of ion channels, neurotransmitter receptors or transporters.Owing to their diversity, more than 50,000 distinct active peptides are theoretically estimated in Conus venoms. These diversified toxins are generally categorized into several superfamilies and/or families based on their characteristic arrangements of cysteine residues and pharmacological actions. Some mechanisms underlying the remarkable diversity of Conus peptides have been postulated: the distinctive gene structure, gene duplication and/or allelic selection, genus speciation, and sophisticated expression pattern and posttranslational modification of these peptides. Due to their highly pharmacological potency and target selectivity, Conus peptides have attracted extensive attention with their potentials to be developed as new research tools in neuroscience field and as novel medications in clinic for pain, epilepsy and other neuropathic disorders. Several instructive lessons for our drug development could be also learnt from these neuropharmacological "expertises". Conus peptides comprise a rich resource for neuropharmacologists, and most of them await to be explored.

  20. C-Peptide and its intracellular signaling.

    Science.gov (United States)

    Hills, Claire E; Brunskill, Nigel J

    2009-01-01

    Although long believed to be inert, C-peptide has now been shown to have definite biological effects both in vitro and in vivo in diabetic animals and in patients with type 1 diabetes. These effects point to a protective action of C-peptide against the development of diabetic microvascular complications. Underpinning these observations is undisputed evidence of C-peptide binding to a variety of cell types at physiologically relevant concentrations, and the downstream stimulation of multiple cell signaling pathways and gene transcription via the activation of numerous transcription factors. These pathways affect such fundamental cellular processes as re-absorptive and/or secretory phenotype, migration, growth, and survival. Whilst the receptor remains to be identified, experimental data points strongly to the existence of a specific G-protein-coupled receptor for C-peptide. Of the cell types studied so far, kidney tubular cells express the highest number of C-peptide binding sites. Accordingly, C-peptide exerts major effects on the function of these cells, and in the context of diabetic nephropathy appears to antagonise the pathophysiological effects of major disease mediators such as TGFbeta1 and TNFalpha. Therefore, based on its cellular activity profile C-peptide appears well positioned for development as a therapeutic tool to treat microvascular complications in type 1 diabetes. PMID:20039003

  1. Novel pH-Sensitive Cyclic Peptides.

    Science.gov (United States)

    Weerakkody, Dhammika; Moshnikova, Anna; El-Sayed, Naglaa Salem; Adochite, Ramona-Cosmina; Slaybaugh, Gregory; Golijanin, Jovana; Tiwari, Rakesh K; Andreev, Oleg A; Parang, Keykavous; Reshetnyak, Yana K

    2016-01-01

    A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue. PMID:27515582

  2. Creating functional peptide architectures at interfaces

    Science.gov (United States)

    Tirrell, Matthew

    2001-03-01

    Short peptide sequences, derived from whole proteins, can be useful synthetic agents for conferring a specific biological function to a material surface. Their ability to do this depends on delivering them to the surface in a biologically recognizable form, that is in a spatial configuration that is not too different from that adopted by the peptide in the whole protein. Most functional proteins have secondary and tertiary levels of structure that are essential to their activities; peptides have simpler but no less important structures. In our work, we have focussed on peptides derived from extracellular matrix proteins. We have found that attaching synthetic lipid tails to peptides fragments gives them two very useful properties for surface modification. The hydrophobic tails give rise to a self-assembly capacity enabling these molecules to organize into membrane, monolayer and bilayer structures. Less expected is that this level of self-assembly induces a second level in the peptide headgroup. Peptides from alpha-helical and triple-helical regions of protein are induced by the lipid tails to form protein-like secondary structures and therefore to have more effective biological activity.

  3. Peptide design for antimicrobial and immunomodulatory applications.

    Science.gov (United States)

    Haney, Evan F; Hancock, Robert E W

    2013-11-01

    The increasing threat of antibiotic resistance in pathogenic bacteria and the dwindling supply of antibiotics available to combat these infections poses a significant threat to human health throughout the world. Antimicrobial peptides (AMPs) have long been touted as the next generation of antibiotics capable of filling the anti-infective void. Unfortunately, peptide-based antibiotics have yet to realize their potential as novel pharmaceuticals, in spite of the immense number of known AMP sequences and our improved understanding of their antibacterial mechanism of action. Recently, the immunomodulatory properties of certain AMPs have become appreciated. The ability of small synthetic peptides to protect against infection in vivo has demonstrated that modulation of the innate immune response is an effective strategy to further develop peptides as novel anti-infectives. This review focuses on the screening methods that have been used to assess novel peptide sequences for their antibacterial and immunomodulatory properties. It will also examine how we have progressed in our ability to identify and optimize peptides with desired biological characteristics and enhanced therapeutic potential. In addition, the current challenges to the development of peptides as anti-infectives are examined and the strategies being used to overcome these issues are discussed.

  4. Immunocytochemical and Immunohistochemical Staining with Peptide Antibodies.

    Science.gov (United States)

    Friis, Tina; Pedersen, Klaus Boberg; Hougaard, David; Houen, Gunnar

    2015-01-01

    Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

  5. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K.

    2008-10-21

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  6. Asymmetric catalysis with short-chain peptides.

    Science.gov (United States)

    Lewandowski, Bartosz; Wennemers, Helma

    2014-10-01

    Within this review article we describe recent developments in asymmetric catalysis with peptides. Numerous peptides have been established in the past two decades that catalyze a wide variety of transformations with high stereoselectivities and yields, as well as broad substrate scope. We highlight here catalytically active peptides, which have addressed challenges that had thus far remained elusive in asymmetric catalysis: enantioselective synthesis of atropoisomers and quaternary stereogenic centers, regioselective transformations of polyfunctional substrates, chemoselective transformations, catalysis in-flow and reactions in aqueous environments.

  7. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells.

    Science.gov (United States)

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  8. Calmodulin of the tropical sea cucumber: Gene structure, inducible expression and contribution to nitric oxide production and pathogen clearance during immune response.

    Science.gov (United States)

    Chen, Ting; Ren, Chunhua; Li, Wuhu; Jiang, Xiao; Xia, Jianjun; Wong, Nai-Kei; Hu, Chaoqun

    2015-08-01

    Calmodulin (CaM) is an essential second messenger protein that transduces calcium signals by binding calcium ions (Ca(2+)) and modulating its interactions with various target proteins. In contrast to vertebrates, where CaM is well established as a cofactor for Ca(2+)-dependent physiological and cellular functions including host defense, there is a paucity of understanding on CaM in invertebrates (such as echinoderms) in response to immune challenge or microbial infections. In this study, we obtained and described the gene sequence of CaM from the tropical sea cucumber Stichopus monotuberculatus, a promising yet poorly characterized aquacultural species. mRNA expression of StmCaM could be detected in the intestine and coelomic fluid after Vibrio alginolyticus injection. Transcriptional and translational expression of StmCaM was inducible in nature, as evidenced by the expression patterns in primary coelomocytes following Vibrio challenge. This response could be mimicked by the Vibrio cells membrane components or lipopolysaccharides (LPS), and blocked by co-treatment of the LPS-neutralizing agent polymyxin B (PMB). Furthermore, inhibition of CaM activity by incubation with its inhibitor trifluoroperazine dihydrochloride (TFP) blunted the production of Vibrio-induced nitric oxide (NO) and augmented the survival of invading Vibrio in coelomocytes. Collectively, our study here supplied the first evidence for echinoderm CaM participation in innate immunity, and provided a functional link between CaM expression and antibacterial NO production in sea cucumber. PMID:25913576

  9. Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Lopez Ferrer, Daniel; Eberlein, P. Elis; Watson, David J.; Squier, Thomas C.

    2009-02-05

    Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance metastasis. In this respect, we have identified a dose-dependent increase in the abundance of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose exposures. Increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX protein complexes associated with DNA repair following macrophage irradiation, with no change in the extent of double-stranded DNA damage. In comparison, when NFκB-dependent pathways are inhibited, through the expression of a dominant-negative IκB construct, there is no significant increase in phosphorylated H2AX upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX mediated DNA-repair pathways is not the result of nonspecific NFκB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation to enhance macrophage radioresistance through processes that include the upregulation of DNA repair pathways involving histone protein H2AX phosphorylation.

  10. Calcium/calmodulin-dependent kinase IV contributes to translation-dependent early synaptic potentiation in the anterior cingulate cortex of adult mice

    Directory of Open Access Journals (Sweden)

    Toyoda Hiroki

    2010-09-01

    Full Text Available Abstract Calcium/calmodulin-dependent kinase IV (CaMKIV phosphorylates the major transcription factor, cyclic AMP-responsive element binding protein (CREB, which plays key roles in synaptic plasticity and memory consolidation. Our previous study showed that long-term potentiation (LTP in the anterior cingulate cortex (ACC was significantly enhanced in transgenic mice overexpressing CaMKIV. Considering that the CaMKIV-CREB pathway plays a central role in the protein synthesis-dependent LTP, it is possible that upregulation of CaMKIV contributes to enhancement of LTP by promoting protein synthesis. To test this possibility, we examined the effects of transcription and translation inhibitors on synaptic potentiation induced by pairing of synaptic activity with postsynaptic depolarization (paired training in ACC pyramidal neurons of wild-type and CaMKIV transgenic mice. We found that synaptic potentiation induced by paired training was partially inhibited by transcription or translation inhibitors both in wild-type and CaMKIV transgenic mice; the extent of inhibition was markedly larger in the CaMKIV transgenic mice than in the wild-type mice. Biochemical and immunohistochemical studies revealed that CaMKIV was distributed in the membrane, cytosol and nucleus of ACC neurons. Our results reveal in the first time a transcription- and translation-dependent component of early synaptic LTP in adult ACC synapses, and demonstrate that CaMKIV enhances early synaptic potentiation by activating new protein synthesis.

  11. Hydrogen sulfide donor sodium hydrosulfide-induced heat tolerance in tobacco (Nicotiana tabacum L) suspension cultured cells and involvement of Ca(2+) and calmodulin.

    Science.gov (United States)

    Li, Zhong-Guang; Gong, Ming; Xie, Hong; Yang, Lan; Li, Jing

    2012-04-01

    Hydrogen sulfide (H(2)S) is considered as a new emerging cell signal in higher plants. Hydrogen sulfide donor, sodium hydrosulfide, pretreatment significantly increased survival percentage of tobacco suspension cultured cells under heat stress and regrowth ability after heat stress, and alleviated decrease in vitality of cells, increase in electrolyte leakage and accumulation of malondialdehyde (MDA). In addition, sodium hydrosulfide-induced heat tolerance was markedly strengthened by application of exogenous Ca(2+) and its ionophore A23187, respectively, while this heat tolerance was weakened by addition of Ca(2+) chelator ethylene glycol-bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), plasma membrane channel blocker La(3+), as well as calmodulin (CaM) antagonists chlorpromazine (CPZ) and trifluoperazine (TFP), respectively, but intracellular channel blocker ruthenium red (RR) did not. These results suggested that sodium hydrosulfide pretreatment could improve heat tolerance in tobacco suspension cultured cells and the acquisition of this heat tolerance requires the entry of extracellular Ca(2+) into cells across the plasma membrane and the mediation of intracellular CaM.

  12. Variants in doublecortin- and calmodulin kinase like 1, a gene up-regulated by BDNF, are associated with memory and general cognitive abilities.

    Directory of Open Access Journals (Sweden)

    Stéphanie Le Hellard

    Full Text Available BACKGROUND: Human memory and general cognitive abilities are complex functions of high heritability and wide variability in the population. The brain-derived neurotrophic factor (BDNF plays an important role in mammalian memory formation. METHODOLOGY / PRINCIPAL FINDING: Based on the identification of genes markedly up-regulated during BDNF-induced synaptic consolidation in the hippocampus, we selected genetic variants that were tested in three independent samples, from Norway and Scotland, of adult individuals examined for cognitive abilities. In all samples, we show that markers in the doublecortin- and calmodulin kinase like 1 (DCLK1 gene, are significantly associated with general cognition (IQ scores and verbal memory function, resisting multiple testing. DCLK1 is a complex gene with multiple transcripts which vary in expression and function. We show that the short variants are all up-regulated after BDNF treatment in the rat hippocampus, and that they are expressed in the adult human brain (mostly in cortices and hippocampus. We demonstrate that several of the associated variants are located in potential alternative promoter- and cis-regulatory elements of the gene and that they affect BDNF-mediated expression of short DCLK1 transcripts in a reporter system. CONCLUSION: These data present DCLK1 as a functionally pertinent gene involved in human memory and cognitive functions.

  13. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells

    Science.gov (United States)

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  14. Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen.

    Science.gov (United States)

    Furuya, Yusui; Denda, Miwako; Sakane, Kyohei; Ogusu, Tomoko; Takahashi, Sumio; Magari, Masaki; Kanayama, Naoki; Morishita, Ryo; Tokumitsu, Hiroshi

    2016-07-01

    To search for novel target(s) of the Ca(2+)-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca(2+)/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca(2+)-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles.

  15. Ca2+/calmodulin-dependent protein kinase II alpha is required for the initiation and maintenance of opioid-induced hyperalgesia.

    Science.gov (United States)

    Chen, Yan; Yang, Cheng; Wang, Zaijie Jim

    2010-01-01

    Repeated administration of opioids not only leads to tolerance and dependence, but also results in nociceptive enhancement called opioid-induced hyperalgesia (OIH). Nociceptive mediators involved in OIH generation remain poorly understood. In the present study, we tested the hypothesis that Ca(2+)/calmodulin-depent protein kinase II (CaMKIIalpha) is critical for OIH. Opioid-induced hyperalgesia was produced by repeated morphine administration or pellet implantation in mice. Correlating with the development of tactile allodynia and thermal hyperalgesia, spinal CaMKIIalpha activity was significantly increased in OIH. KN93, a CaMKII inhibitor, dose- and time-dependently reversed OIH and CaMKII activation without impairing locomotor coordination. To elucidate the specific CaMKII isoform involved, we targeted CaMKIIalpha by using small interfering RNA and demonstrated that knockdown of spinal CaMKIIalpha attenuated OIH. Furthermore, morphine failed to induce OIH in CaMKIIalpha(T286A) point mutant mice, although wild-type littermate mice developed robust OIH after repeated treatments with morphine. These data implicate, for the first time, an essential role of CaMKIIalpha as a cellular mechanism leading to and maintaining opioid-induced hyperalgesia.

  16. The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization.

    Science.gov (United States)

    Licata, Stephanie C; Pierce, R Christopher

    2003-04-01

    Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. PMID:12641723

  17. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  18. Competitive binding of postsynaptic density 95 and Ca2+-calmodulin dependent protein kinase Ⅱ to N-methyl-D-aspartate receptor subunit 2B in rat brain

    Institute of Scientific and Technical Information of China (English)

    Fan-jie MENG; Jun GUO; Bo SONG; Xue-bo YAN; Guang-yi ZHANG

    2004-01-01

    AIM: To investigate the interactions among postsynaptic density 95 (PSD-95), Ca2+-calmodulin dependent protein kinase Ⅱα (CaMKⅡα), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats. METHODS: Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKⅡα to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKⅡ) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins. RESULTS: The alternations of the binding level of PSD-95 and CaMKⅡα to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10 min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKⅡα to NR2B. CONCLUSION: PSD-95 competes with CaMKⅡ to bind to NR2B during ischemia and reperfusion in rat hippocampus.

  19. Apo-states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain

    Science.gov (United States)

    Findeisen, Felix; Rumpf, Christine; Minor, Daniel L.

    2013-01-01

    In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation (CDI) and limits calcium entry, whereas CaBP1 blocks CDI and allows sustained calcium influx. Here, we combine isothermal titration calorimetry (ITC) with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium binding properties. The observation that the apo-forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. PMID:23811053

  20. Peptides from milk proteins and their properties.

    Science.gov (United States)

    Kilara, Arun; Panyam, Dinakar

    2003-01-01

    This review has attempted to study the literature pertaining to peptides derived from milk proteins. Hydrolysis of milk proteins to generate peptides has been practiced for a long time and it was recognized early on in this process that the taste of hydrolyzates might hinder use of these products in food formulations. Modification of protein is necessary to form a more acceptable or utilizable product, to form a product that is less susceptible to deteriorative reactions and to form a product that is of higher nutritionall quality. Modifications may be achieved by a number of chemical and enzymatic means. This review has considered only enzymatic modification of dairy proteins. Modified proteins contain peptides and some of these peptides have been purified and their functionalities have been compared with unmodified proteins. This paper has examined the literature pertaining to improvement in functionality of enzyme-modified proteins. Improvements in solubility, emulsification, foaming and gelation were examined. There is limited information available on the sequence of the peptides necessary to improve the functional characteristics of proteins. Knowing the sequences of desirable functional peptides can lead to genetic alteration of proteins to improve functionality. Addition of synthetic peptides to intact proteins may be another way in which the functionality of proteins can be augmented. Some of the peptides in milk proteins are capable of affecting biological functions of an organism. These effects can be antimicrobial and probiotic, i.e., prevent the growth and proliferation of undesirable and pathogenic organisms, or they may promote the growth of desirable bacteria in the digestive tract of humans and animals. Peptides derived from milk protein have been shown to exert digestive and metabolic effects as well. They may also influence the immune system. These biological effects may play an important role in the development of medical foods that treat or

  1. Screening of a specific peptide binding to VPAC1 receptor from a phage display peptide library.

    Directory of Open Access Journals (Sweden)

    Bo Tang

    Full Text Available BACKGROUND/PURPOSE: The VPAC1 receptor, a member of the vasoactive intestinal peptide receptors (VIPRs, is overexpressed in the most frequently occurring malignant tumors and plays a major role in the progression and angiogenesis of a number of malignancies. Recently, phage display has become widely used for many applications, including ligand generation for targeted imaging, drug delivery and therapy. In this work, we developed a panning procedure using a phage display peptide library to select a peptide that specifically binds to the VPAC1 receptor to develop a novel targeted probe for molecular imaging and therapy. METHODS: CHO-K1 cells stably expressing VPAC1 receptors (CHO-K1/VPAC1 cells were used to select a VPAC1-binding peptide from a 12-mer phage peptide library. DNA sequencing and homologous analysis of the randomly selected phage clones were performed. A cellular ELISA was used to determine the most selectively binding peptide for further investigation. Binding specificity to the VPAC1 receptor was analyzed by competitive inhibition ELISA and flow cytometry. The binding ability of the selected peptide to CHO-K1/VPAC1 cells and colorectal cancer (CRC cell lines was confirmed using fluorescence microscopy and flow cytometry. RESULTS: A significant enrichment of phages that specifically bound to CHO-K1/VPAC1 cells was obtained after four rounds of panning. Of the selected phage clones, 16 out of 60 shared the same peptide sequence, GFRFGALHEYNS, which we termed the VP2 peptide. VP2 and vasoactive intestinal peptide (VIP competitively bound to the VPAC1 receptor. More importantly, we confirmed that VP2 specifically bound to CHO-K1/VPAC1 cells and several CRC cell lines. CONCLUSION: Our results demonstrate that the VP2 peptide could specifically bind to VPAC1 receptor and several CRC cell lines. And VP2 peptide may be a potential candidate to be developed as a useful diagnostic molecular imaging probe for early detection of CRC.

  2. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    OpenAIRE

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and functi...

  3. Collagen-like peptides and peptide-polymer conjugates in the design of assembled materials

    OpenAIRE

    Luo, Tianzhi; Kiick, Kristi L.

    2013-01-01

    Collagen is the most abundant protein in mammals, and there has been long-standing interest in understanding and controlling collagen assembly in the design of new materials. Collagen-like peptides (CLP), also known as collagen-mimetic peptides (CMP) or collagen-related peptides (CRP), have thus been widely used to elucidate collagen triple helix structure as well as to produce higher-order structures that mimic natural collagen fibers. This mini-review provides an overview of recent progress...

  4. A statistical approach to determining responses to individual peptides from pooled-peptide ELISpot data.

    Science.gov (United States)

    Ström, Peter; Støer, Nathalie; Borthwick, Nicola; Dong, Tao; Hanke, Tomáš; Reilly, Marie

    2016-08-01

    To investigate in detail the effect of infection or vaccination on the human immune system, ELISpot assays are used to simultaneously test the immune response to a large number of peptides of interest. Scientists commonly use "peptide pools", where, instead of an individual peptide, a test well contains a group of peptides. Since the response from a well may be due to any or many of the peptides in the pool, pooled assays usually need to be followed by confirmatory assays of a number of individual peptides. We present a statistical method that enables estimation of individual peptide responses from pool responses using the Expectation Maximization (EM) algorithm for "incomplete data". We demonstrate the accuracy and precision of these estimates in simulation studies of ELISpot plates with 90 pools of 6 or 7 peptides arranged in three dimensions and three Mock wells for the estimation of background. In analysis of real pooled data from 6 subjects in a HIV-1 vaccine trial, where 199 peptides were arranged in 80 pools if size 9 or 10, our estimates were in very good agreement with the results from individual-peptide confirmatory assays. Compared to the classical approach, we could identify almost all the same peptides with high or moderate response, with less than half the number of confirmatory tests. Our method facilitates efficient use of the information available in pooled ELISpot data to avoid or reduce the need for confirmatory testing. We provide an easy-to-use free online application for implementing the method, where on uploading two spreadsheets with the pool design and pool responses, the user obtains the estimates of the individual peptide responses. PMID:27196788

  5. Peptide Internalization Enabled by Folding: Triple Helical Cell-Penetrating Peptides

    OpenAIRE

    Shinde, Aparna; Feher, Katie M.; Hu, Chloe; Slowinska, Katarzyna

    2014-01-01

    Cell-Penetrating Peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degrada...

  6. Determination of peptide content and purity of DOTA-peptides by metal ion titration and UPLC. An alternative method to monitor quality of DOTA-peptides

    International Nuclear Information System (INIS)

    PRRT requires high specific activities, thus at low molar ratio between DOTA-peptide and radioactivity. Therefore, the ingredients of the reaction, including (radio)metals and DOTA-peptide must be pure and the content known. Our aim was to quantify content and purity of DOTA-peptide by a base-to-base separation of DOTA-peptide and metal-DOTA-peptide by UPLC and UV-detection. Quantification of these peaks reveals an accurate and sensitive method to quantify purity and content of DOTA-peptides. Moreover, this technique enables monitoring of the (radio)labeling process and co-introduction of impurities, including metal ions. (author)

  7. Charge Transport Phenomena in Peptide Molecular Junctions

    Directory of Open Access Journals (Sweden)

    Alessandra Luchini

    2008-01-01

    Full Text Available Inelastic electron tunneling spectroscopy (IETS is a valuable in situ spectroscopic analysis technique that provides a direct portrait of the electron transport properties of a molecular species. In the past, IETS has been applied to small molecules. Using self-assembled nanoelectronic junctions, IETS was performed for the first time on a large polypeptide protein peptide in the phosphorylated and native form, yielding interpretable spectra. A reproducible 10-fold shift of the I/V characteristics of the peptide was observed upon phosphorylation. Phosphorylation can be utilized as a site-specific modification to alter peptide structure and thereby influence electron transport in peptide molecular junctions. It is envisioned that kinases and phosphatases may be used to create tunable systems for molecular electronics applications, such as biosensors and memory devices.

  8. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.;

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  9. Ribosomally synthesized peptides from natural sources.

    Science.gov (United States)

    Singh, Nidhi; Abraham, Jayanthi

    2014-04-01

    There are many antibiotic-resistant microbial pathogens that have emerged in recent years causing normal infections to become harder and sometimes impossible to treat. The major mechanisms of acquired resistance are the ability of the microorganisms to destroy or modify the drug, alter the drug target, reduce uptake or increase efflux of the drug and replace the metabolic step targeted by the drug. However, in recent years, resistant strains have been reported from almost every environment. New antimicrobial compounds are of major importance because of the growing problem of bacterial resistance, and antimicrobial peptides have been gaining a lot of interest. Their mechanism of action, however, is often obscure. Antimicrobial peptides are widespread and have a major role in innate immunity. An increasing number of peptides capable of inhibiting microbial growth are being reviewed here. In this article, we consider the possible use of antimicrobial peptides against pathogens.

  10. Evolution of Antimicrobial Peptides to Self-Assembled Peptides for Biomaterial Applications

    Directory of Open Access Journals (Sweden)

    Alice P. McCloskey

    2014-10-01

    Full Text Available Biomaterial-related infections are a persistent burden on patient health, recovery, mortality and healthcare budgets. Self-assembled antimicrobial peptides have evolved from the area of antimicrobial peptides. Peptides serve as important weapons in nature, and increasingly medicine, for combating microbial infection and biofilms. Self-assembled peptides harness a “bottom-up” approach, whereby the primary peptide sequence may be modified with natural and unnatural amino acids to produce an inherently antimicrobial hydrogel. Gelation may be tailored to occur in the presence of physiological and infective indicators (e.g. pH, enzymes and therefore allow local, targeted antimicrobial therapy at the site of infection. Peptides demonstrate inherent biocompatibility, antimicrobial activity, biodegradability and numerous functional groups. They are therefore prime candidates for the production of polymeric molecules that have the potential to be conjugated to biomaterials with precision. Non-native chemistries and functional groups are easily incorporated into the peptide backbone allowing peptide hydrogels to be tailored to specific functional requirements. This article reviews an area of increasing interest, namely self-assembled peptides and their potential therapeutic applications as innovative hydrogels and biomaterials in the prevention of biofilm-related infection.

  11. From a pro-apoptotic peptide to a lytic peptide: One single residue mutation.

    Science.gov (United States)

    Zhou, Xi-Rui; Zhang, Qiang; Tian, Xi-Bo; Cao, Yi-Meng; Liu, Zhu-Qing; Fan, Ruru; Ding, Xiu-Fang; Zhu, Zhentai; Chen, Long; Luo, Shi-Zhong

    2016-08-01

    Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys→Leu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics. PMID:27207743

  12. Gene Transfer with Poly-Melittin Peptides

    OpenAIRE

    Chen, Chang-Po; Kim, Ji-Seon; Steenblock, Erin; Liu, Dijie; Rice, Kevin G.

    2006-01-01

    The 26 amino acid hemolytic melittin peptide was converted into a gene transfer peptide that binds to DNA and polymerized through disulfide bond formation. Melittin analogues were synthesized by addition of one to four Lys repeats at either the C or N-subterminal end along with terminal Cys residues. Melittin analogues were able to bind and polymerize on plasmids resulting in the formation of DNA condensates. In the absence of DNA, melittin analogues retained their red blood cell hemolytic po...

  13. Bioactive peptides and proteins in disease

    OpenAIRE

    Refai, Essam

    2004-01-01

    Regulatory peptides and marker proteins are important to study in order to understand disease mechanisms. This applies of course also to our common diseases where all relationships are not yet known. Cancer and diabetes are two such complex diseases that affect hundreds of millions of people worldwide. This thesis addresses particular aspects of these two diseases, regarding one regulatory peptide (VIP, vasoactive intestinal polypeptide) that may be useful for tumor tracing ...

  14. Natriuretic peptides, obesity and cardiovascular diseases

    Directory of Open Access Journals (Sweden)

    Yaniel Castro-Torres

    2015-02-01

    Full Text Available Obesity, hypertension and heart failure are conditions commonly associated with each other. Recent investigations have demonstrated that low plasmatic levels of natriuretic peptides are linked with obesity. Thus, knowing the actions of these hormones in water and salt homeostasis, it is possible to establish that low levels of natriuretic peptides may be the common denominator among obesity, hypertension and heart failure. Knowledge on this topic is crucial to develop further investigation for definitive conclusions.

  15. Dietary fiber, gut peptides, and adipocytokines

    OpenAIRE

    Sánchez, David; Miguel, Marta; Aleixandre, Amaya

    2012-01-01

    The consumption of dietary fiber (DF) has increased since it was related to the prevention of a range of illnesses and pathological conditions. DF can modify some gut hormones that regulate satiety and energy intake, thus also affecting lipid metabolism and energy expenditure. Among these gut hormones are ghrelin, glucagon-like peptide 1, peptide YY, and cholecystokinin. Adipose tissue is known to express and secrete a variety of products known as >adipocytokines,> which are also affected by ...

  16. Peptide oligomers for holographic data storage

    DEFF Research Database (Denmark)

    Berg, Rolf Henrik; Hvilsted, Søren; Ramanujam, P.S.

    1996-01-01

    SEVERAL classes of organic materials (such as photoanisotropic liquid-crystalline polymers(1-4) and photorefractive polymers(5-7)) are being investigated for the development of media for optical data storage. Here we describe a new family of organic materials-peptide oligomers containing azobenze....... Straightforward extension of this peptide-based strategy to other molecular structures should allow the rational design of a wide range of organic materials with potentially useful optical properties....

  17. From antimicrobial to anticancer peptides. A review.

    OpenAIRE

    Diana eGaspar; A. Salomé eVeiga; Miguel A.R.B. eCastanho

    2013-01-01

    Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms. Although AMPs have been essentially studied and developed as potential alternatives for fighting infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been regarded as a therapeutic strategy to explore. As human cancer remains a cause of high morbidity and mortality worldwide, an urgent need of new, selective...

  18. Liquid-phase synthesis of bridged peptides using olefin metathesis of a protected peptide with a long aliphatic chain anchor.

    Science.gov (United States)

    Aihara, Keisuke; Komiya, Chiaki; Shigenaga, Akira; Inokuma, Tsubasa; Takahashi, Daisuke; Otaka, Akira

    2015-02-01

    Bridged peptides including stapled peptides are attractive tools for regulating protein-protein interactions (PPIs). An effective synthetic methodology in a heterogeneous system for the preparation of these peptides using olefin metathesis and hydrogenation of protected peptides with a long aliphatic chain anchor is reported.

  19. Affinity-based release of polymer-binding peptides from hydrogels with the target segments of peptides.

    Science.gov (United States)

    Serizawa, Takeshi; Fukuta, Hiroki; Date, Takaaki; Sawada, Toshiki

    2016-02-01

    Peptides with affinities for the target segments of polymer hydrogels were identified by biological screening using phage-displayed peptide libraries, and these peptides exhibited an affinity-based release capability from hydrogels. The results from cell culture assays demonstrated the sustained anticancer effects of the drug-conjugated peptides that were released from the hydrogels.

  20. Membrane manufacture for peptide separations

    KAUST Repository

    Kim, DooLi

    2016-06-07

    Nanostructured polymeric membranes are key tools in biomedical applications such as hemodialysis, protein separations, in the food industry, and drinking water supply from seawater. Despite of the success in different separation processes, membrane manufacture itself is at risk, since the most used solvents are about to be banned in many countries due to environmental and health issues. We propose for the first time the preparation of polyethersulfone membranes based on dissolution in the ionic liquid 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DEP). We obtained a series of membranes tailored for separation of solutes with molecular weight of 30, 5, 1.3, and 1.25 kg mol-1 with respective water permeances of 140, 65, 30 and 20 Lm-2h-1bar-1. We demonstrate their superior efficiency in the separation of complex mixtures of peptides with molecular weights in the range of 800 to 3500 gmol-1. Furthermore, the thermodynamics and kinetics of phase separation leading to the pore formation in the membranes were investigated. The rheology of the solutions and the morphology of the prepared membranes were examed and compared to those of polyethersulfone in organic solvents currently used for membrane manufacture.