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1.
Diamide insecticides selectively activate insect ryanodine receptors (RyRs), inducing uncontrolled release of calcium ions, and causing muscle contraction, paralysis and eventually death. The RyRG4946E substitution associated with diamide resistance has been identified in three lepidopteran pests, Plutella xylostella, Tuta absoluta and Chilo suppressalis. Recently, the T. absoluta RyRG4946V mutation was knocked into the model insect Drosophila melanogaster by CRISPR/Cas9 mediated genome editing and provided in vivo functional confirmation for its role in diamide resistance. In the present study, we successfully introduced the RyRG4946E mutation with CRISPR/Cas9 technology into a lepidopteran pest of global importance, Spodoptera exigua. The genome-edited strain (named 4946E) homozygous for the SeRyRG4946E mutation exhibited 223-, 336- and >1000-fold resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively when compared to the wild type strain (WHS) of S. exigua. Reciprocal crossing experiments revealed that the target-site resistance in strain 4946E underlies an autosomal and almost recessive mode of inheritance for anthranilic diamides, whereas it was completely recessive for flubendiamide. Our results not only provided in vivo functional validation of the RyRG4946E mutation in conferring high levels of resistance to diamide insecticides for the first time in a controlled genetic background of a lepidopteran pest, but also revealed slight differences on the level of resistance between anthranilic diamides (chlorantraniliprole and cyantraniliprole) and flubendiamide conferred by the SeRyRG4946E mutation. 相似文献
2.
Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains 总被引:2,自引:0,他引:2
Ca2+ and calmodulin (CaM), a key Ca2+ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca2+ and CaM in defense signaling, we used 35S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound 35S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca2+ whereas EGTA, a Ca2+ chelator, abolished binding, confirming that PICBP binds CaM in a Ca2+-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsis
accelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca2+-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca2+-dependent CaM-binding domains. 相似文献
3.
Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution 总被引:4,自引:0,他引:4
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. 相似文献
4.
Diamide insecticides are used widely against lepidopteran pests, acting as potent activators of insect Ryanodine Receptors (RyRs) and thus inducing muscle contraction and eventually death. However, resistant phenotypes have recently evolved in the field, associated with the emergence of target site resistance mutations (G4946E/V and I4790M). We investigated the frequency of the mutations found in a resistant population of Tuta absoluta from Greece (G4946V ~79% and I4790M ~21%) and the associated diamide resistance profile: there are very high levels of resistance against chlorantraniliprole (9329-fold) and flubendiamide (4969-fold), but moderate levels against cyantraniliprole (191-fold). To further investigate functionally the contribution of each mutation in the resistant phenotype, we used CRISPR/Cas9 to generate genome modified Drosophila carrying alternative allele combinations, and performed toxicity bioassays against all three diamides. Genome modified flies bearing the G4946V mutation exhibited high resistance ratios to flubendiamide (91.3-fold) and chlorantraniliprole (194.7-fold) when compared to cyantraniliprole (5.4-fold). Flies naturally wildtype for the I4790M mutation were moderately resistant to flubendiamide (15.3-fold) but significantly less resistant to chlorantraniliprole (7.5-fold), and cyantraniliprole (2.3-fold). These findings provide in vivo functional genetic confirmation for the role and relative contribution of RyR mutations in diamide resistance and suggest that the mutations confer subtle differences on the relative binding affinities of the three diamides at an overlapping binding site on the RyR protein. 相似文献
5.
Yong Tao Steven Gutteridge Eric A. Benner Lihong Wu Daniel F. Rhoades Matthew D. Sacher Michel A. Rivera Johan Desaeger Daniel Cordova 《Insect biochemistry and molecular biology》2013,43(9):820-828
Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors. 相似文献
6.
Pei-Feng Chen 《Archives of biochemistry and biophysics》2009,486(2):132-140
Human endothelial nitric-oxide synthase (eNOS) is a complex enzyme, requiring binding of calmodulin (CaM) for electron transfer. The prevailing view is that calcium-activated CaM binds eNOS at the canonical binding site located at residues 493-510, which induces a conformational change to facilitate electron transfer. Here we demonstrated that the CaM enhances the rate of electron transfer from NADPH to FAD on a truncated eNOS FAD subdomain (residues 682-1204) purified from baculovirus-infected Sf9 cells, suggesting more complicated regulatory mechanism of CaM on eNOS. Metabolically 35S-labeled CaM overlay on fusion proteins spanning the entire linear sequence of eNOS revealed three positive 35S-CaM binding fragments: sequence 66-205, sequence 460-592, and sequence 505-759. Synthetic peptides derived from these fragments are tested for their effects on CaM binding and eNOS catalytic activities. Peptides corresponding to the proximal heme-binding site (E1, residues 174-193) and the CD1 linker connecting FAD/FMN subdomains (E4, residues 729-757) bind CaM at both high Ca2+ (Ca2+CaM) and low Ca2+ (apoCaM) concentrations, whereas peptide of the canonical CaM-binding helix (E2, residues 493-510) binds only Ca2+CaM. All three peptides E1, E2 and E4 significantly inhibit oxygenase activity in a concentration-dependent manner, but only E2 effectively inhibits reductase activity. Concurrent experiments with human iNOS showed major differences in the CaM binding properties between eNOS and iNOS. The results suggest that multiple regions of eNOS might interact with CaM with differential Ca2+ sensitivity in vivo. A possible mechanism in regulating eNOS activation and deactivation is proposed. 相似文献
7.
Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores 总被引:1,自引:0,他引:1
Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca2+ by pretreatment with the cell-permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells. 相似文献
8.
Toxin(s) from the ichthyotoxic red tide alga Heterosigma akashiwo have been responsible for the destruction of millions of dollars of finfish aquaculture around the globe. Mechanisms of toxicity may include the production of reactive oxygen species (ROS) or organic toxins. The purpose of this study was to investigate the bioactivity of extracellular organic compounds from cultures of H. akashiwo. Cytosolic free calcium levels ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells infected with baculoviruses encoding the M1 muscarinic receptor were monitored.Exposure of cells to Heterosigma organics increased [Ca2+]i up to 120 nM above basal levels (two-fold increase). Within minutes following exposure of the cells to the organics, the increase in [Ca2+]i peaked and was followed by a slightly reduced, yet sustained plateau. This plateau was maintained for the duration of an experiment (>15 min) and was inhibitable by lanthanum. Furthermore, stimulation of Ca2+ release from intracellular stores by carbachol (muscarinic agonist) or thapsigargin (sarco-endoplasmic reticulum Ca2+-ATPases, SERCA inhibitor) potentiated the [Ca2+]i response induced by the organics resulting in a maximal increase of >250 nM above basal levels (three-fold increase). However, the [Ca2+]i response to Heterosigma organics was strictly dependent on the presence of extracellular calcium. Flow cytometric analyses revealed that these organics induced apoptosis of these same cells. Collectively, our data indicate that extracellular organics from cultures of H. akashiwo acutely increase [Ca2+]i in cells by inhibiting the plasma membrane Ca2+-ATPase transporter and ultimately induce apoptotic cell death. These organics may play a significant role in the ichthyotoxic and allelopathic behaviour of this alga. 相似文献
9.
Kalina Dimova Noa Lipstein Donghan Lee Stefan Becker Nils Brose Olaf Jahn Christian Griesinger 《The EMBO journal》2010,29(3):680-691
Ca2+ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca2+–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca2+ signals. We solved the structure of Ca2+4–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca2+2–CaM complex. The Ca2+ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca2+–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca2+]i, whereas the N‐module acts as a sensor at micromolar [Ca2+]i. This Ca2+/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca2+‐dependent modulation of short‐term synaptic plasticity. 相似文献
10.
It is now generally accepted that non‐genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca2+ is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ extrusion in membranes incubated with 17‐β‐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. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
11.
Thomas P. Selby George P. Lahm Thomas M. Stevenson Kenneth A. Hughes Daniel Cordova I. Billy Annan James D. Barry Eric A. Benner Martin J. Currie Thomas F. Pahutski 《Bioorganic & medicinal chemistry letters》2013,23(23):6341-6345
Anthranilic diamides are an exceptionally active class of insect control chemistry that selectively activates insect ryanodine receptors causing mortality from uncontrolled release of calcium ion stores in muscle cells. Work in this area led to the successful commercialization of chlorantraniliprole for control of Lepidoptera and other insect pests at very low application rates. In search of lower log P analogs with improved plant systemic properties, exploration of cyano-substituted anthranilic diamides culminated in the discovery of a second product candidate, cyantraniliprole, having excellent activity against a wide range of pests from multiple insect orders. Here we report on the chemistry, biology and structure–activity trends for a series of cyanoanthranilic diamides from which cyantraniliprole was selected for commercial development. 相似文献
12.
Eugene Emmer Richard P. Rood John H. Wesolek Michael E. Cohen R. Scott Braithwaite Geoffrey W. G. Sharp Heini Murer Mark Donowitz 《The Journal of membrane biology》1989,108(3):207-215
Summary In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl– absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45±13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on theV
max and not on theK
m
for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120nm. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. we conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption. 相似文献
13.
Wataru Nunomura Noriyoshi Isozumi Shigeyoshi Nakamura Yuji Jinbo Shinya Ohki Shun-ichi Kidokoro Hideki Wakui Yuichi Takakuwa 《Cell biochemistry and biophysics》2014,69(1):7-19
Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca2+ but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca2+-dependent manner. We have previously mapped a Ca2+-independent CaM-binding site, pep11 (A264KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca2+ (Ca2+/CaM), interacts simultaneously with pep11 and with Ser185 in A181KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca2+/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca2+/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca2+. Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca2+. Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca2+/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca2+/CaM-dependent regulation of R30 binding to membrane proteins. 相似文献
14.
Kwangwoon Lee Eric A. Kumar Kevin N. Dalby Ranajeet Ghose 《Protein science : a publication of the Protein Society》2019,28(12):2089-2098
Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+, CaM exclusively utilizes its C‐terminal lobe (CaMC) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+‐loaded N‐terminal lobe of CaM (CaMN) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+‐free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex. 相似文献
15.
Xu Wang Liang Wen Xiong Amina El Ayadi Darren Boehning John A. Putkey 《The Journal of biological chemistry》2013,288(3):2040-2048
PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca2+ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca2+ binding activity, which may modulate Ca2+ binding to CaM by stabilizing an initial Ca2+-CaM complex or by electrostatically steering Ca2+ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca2+ dynamics, we tested the hypothesis that it influences ligand-dependent Ca2+ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca2+ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca2+ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca2+ binding to CaM greatly inhibit its effect on ATP-induced Ca2+ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca2+ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling. 相似文献
16.
17.
Martin González‐Andrade Rachel Mata Abraham Madariaga‐Mazón Rogelio Rodríguez‐Sotres Luis del Pozo‐Yauner Alejandro Sosa‐Peinado 《Journal of molecular recognition : JMR》2013,26(4):165-174
Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca2+–CaM, Ca2+–CaM–PDE1A, and Ca2+–CaM–MLCK complexes, CPZ apparent dissociation constants (Kds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca2+–hCaM M124C–mBBr quenched the fluorescence (Kd = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca2+–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca2+–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca2+–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
18.
Elke De Vuyst Nan Wang Elke Decrock Marijke De Bock Mathieu Vinken Marijke Van Moorhem Charles Lai Maxime Culot Vera Rogiers Romeo Cecchelli Christian C. Naus W. Howard Evans Luc Leybaert 《Cell calcium》2009,46(3):176-187
Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca2+ ([Ca2+]i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca2+]i was in the 500 nM range but the responses disappeared with larger [Ca2+]i transients. Ca2+-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca2+-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca2+]i triggers hemichannel opening, not by a direct action of Ca2+ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca2+]i and acting to restrain cellular ATP loss. 相似文献
19.
Silviya R. Stateva Valentina Salas Estefanía Anguita Gustavo Benaim Antonio Villalobo 《PloS one》2015,10(6)
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. 相似文献
20.
D. Brent Halling Sophia A. Kenrick Austen F. Riggs Richard W. Aldrich 《The Journal of general physiology》2014,143(2):231-252
Ca2+ activates SK Ca2+-activated K+ channels through the protein Ca2+ sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca2+ regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca2+ concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca2+, SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca2+, 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca2+, the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca2+ and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca2+ or with CaM in molar excess. In low Ca2+ both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca2+. These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating. 相似文献