The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Hillel Fromm - One of the best experts on this subject based on the ideXlab platform.
-
Calmodulin Binding transcription activator camta 3 mediates biotic defense responses in arabidopsis
FEBS Letters, 2008Co-Authors: Yael Galon, Roy Nave, Joy M Boyce, Dikla Nachmias, Marc R Knight, Hillel FrommAbstract: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.
-
camtas Calmodulin Binding transcription activators from plants to human
FEBS Letters, 2007Co-Authors: Aliza Finkler, Ruth Asherypadan, Hillel FrommAbstract:Recently, a novel family of Calmodulin-Binding transcription activators (CAMTAs) was reported in various eukaryotes. All CAMTAs share a similar domain organization, with a novel type of sequence-specific DNA-Binding domain (designated CG-1). This domain could bind DNA directly and activate transcription, or interact with other transcription factors, not through DNA Binding, thus acting as a co-activator of transcription. Investigations of CAMTAs in various organisms imply a broad range of functions from sensory mechanisms to embryo development and growth control, highlighted by the apparent involvement of mammalian CAMTA2 in cardiac growth, and of CAMTA1 in tumor suppression and memory performance.
-
A novel family of Calmodulin-Binding transcription activators in multicellular organisms
Journal of Biological Chemistry, 2002Co-Authors: Nicolas Bouche, Ariel Scharlat, Wayne Snedden, David Bouchez, Hillel FrommAbstract:Screening of cDNA expression libraries derived from plants exposed to stress, with35S-labeled recombinant Calmodulin as a probe, revealed a new family of proteins containing a transcription activation domain and two types of DNA-Binding domains designated the CG-1 domain and the transcription factor immunoglobulin domain, ankyrin repeats, and a varying number of IQ Calmodulin-Binding motifs. Based on domain organization and amino acid sequence comparisons, similar proteins, with the same domain organization, were identified in the genomes of other multicellular organisms including human,Drosophila, and Caenorhabditis, whereas none were found in the complete genomes of single cell eukaryotes and prokaryotes. This family of proteins was designated Calmodulin-Binding transcription activators (CAMTAs). Arabidopsis thalianacontains six CAMTA genes (AtCAMTA1–AtCAMTA6). The transcription activation domain of AtCAMTA1 was mapped by testing a series of protein fusions with the DNA-Binding domain of the bacterial LexA transcription factor and two reporter genes fused to LexA recognition sequences in yeast cells. Two human proteins designated HsCAMTA1 and HsCAMTA2 were also shown to activate transcription in yeast using the same reporter system. Subcellular fractionation ofArabidopsis tissues revealed the presence of CAMTAs predominantly in the nucleus. Calmodulin Binding assays identified a region of 25 amino acids capable of Binding Calmodulin with high affinity (K d = 1.2 nm) in the presence of calcium. We suggest that CAMTAs comprise a conserved family of transcription factors in a wide range of multicellular eukaryotes, which possibly respond to calcium signaling by direct Binding of Calmodulin.
-
a high affinity Calmodulin Binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide Binding domains
Plant Molecular Biology, 2000Co-Authors: Tzahi Arazi, Boaz Kaplan, Hillel FrommAbstract:Recently we isolated a cDNA encoding a tobacco plasma membrane Calmodulin-Binding channel protein (designated NtCBP4) with a putative cyclic nucleotide-Binding domain. Here we analyzed in detail the interaction of NtCBP4 with Calmodulin. A full-length recombinant NtCBP4 (81 kDa) expressed in Sf9 insect cells, and the corresponding tobacco membrane protein were solubilized from their respective membrane fractions and partially purified by Calmodulin affinity chromatography. NtCBP4 was detected in the eluted fractions using specific antibodies raised against the recombinant protein. By Binding [35S]-Calmodulin to recombinant NtCBP4 truncations fused to glutathione S-transferase, we identified a single region consisting of 66 amino acids capable of Binding Calmodulin. A 23 amino acid synthetic peptide from within this region formed a complex with Calmodulin in the presence of calcium. We measured the fluorescence of dansyl-Calmodulin interacting with this peptide, which revealed a dissociation constant of about 8 nM. The NtCBP4 Calmodulin-Binding domain was found to perfectly coincide with a phylogenetically conserved αC-helix motif of its putative cyclic nucleotide-Binding domain. Furthermore, a 23 amino acid region in an equivalent site in the cAMP-Binding domain of a mammalian protein kinase regulatory subunit was also found to bind Calmodulin. Thus, coinciding Calmodulin- and cyclic nucleotide-Binding domains may serve as a point of communication between calcium and cyclic nucleotide signal transduction pathways in plants and animals.
-
a tobacco plasma membrane Calmodulin Binding transporter confers ni2 tolerance and pb2 hypersensitivity in transgenic plants
Plant Journal, 1999Co-Authors: Tzahi Arazi, Boaz Kaplan, Ramanjulu Sunkar, Hillel FrommAbstract:All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal-uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant Calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum Calmodulin-Binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K+ and to non-selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two-phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with reduced Ni2+ accumulation and enhanced Pb2+ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb2+. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.
Danton H Oday - One of the best experts on this subject based on the ideXlab platform.
-
Calmodulin Binding proteins and alzheimer s disease biomarkers regulatory enzymes and receptors that are regulated by Calmodulin
International Journal of Molecular Sciences, 2020Co-Authors: Danton H OdayAbstract:The integral role of Calmodulin in the amyloid pathway and neurofibrillary tangle formation in Alzheimer's disease was first established leading to the "Calmodulin Hypothesis". Continued research has extended our insight into the central function of the small calcium sensor and effector Calmodulin and its target proteins in a multitude of other events associated with the onset and progression of this devastating neurodegenerative disease. Calmodulin's involvement in the contrasting roles of calcium/CaM-dependent kinase II (CaMKII) and calcineurin (CaN) in long term potentiation and depression, respectively, and memory impairment and neurodegeneration are updated. The functions of the proposed neuronal biomarker neurogranin, a Calmodulin Binding protein also involved in long term potentiation and depression, is detailed. In addition, new discoveries into Calmodulin's role in regulating glutamate receptors (mGluR, NMDAR) are overviewed. The interplay between Calmodulin and amyloid beta in the regulation of PMCA and ryanodine receptors are prime examples of how the buildup of classic biomarkers can underly the signs and symptoms of Alzheimer's. The role of Calmodulin in the function of stromal interaction molecule 2 (STIM2) and adenosine A2A receptor, two other proteins linked to neurodegenerative events, is discussed. Prior to concluding, an analysis of how targeting Calmodulin and its Binding proteins are viable routes for Alzheimer's therapy is presented. In total, Calmodulin and its Binding proteins are further revealed to be central to the onset and progression of Alzheimer's disease.
-
nucleolar localization and identification of nuclear nucleolar localization signals of the Calmodulin Binding protein nucleomorphin during growth and mitosis in dictyostelium
Histochemistry and Cell Biology, 2011Co-Authors: Andrew Catalano, Danton H OdayAbstract:The Calmodulin-Binding protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and Calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar protein. Prior to this study, the dynamics of specific nucleolar proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar protein and the only nucleolar Calmodulin-Binding protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.
-
differentiation inducing factor 1 dif 1 induces gene and protein expression of the dictyostelium nuclear Calmodulin Binding protein nucleomorphin
Cellular Signalling, 2009Co-Authors: Danton H Oday, Yekaterina Poloz, Michael A MyreAbstract:Abstract 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.
-
isolation characterization and bioinformatic analysis of Calmodulin Binding protein cmbb reveals a novel tandem ip22 repeat common to many dictyostelium and mimivirus proteins
Biochemical and Biophysical Research Communications, 2006Co-Authors: Danton H Oday, Michael A Myre, Munmun Chatterjeechakraborty, Karsten Suhre, Sara E ChavezAbstract:Abstract 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 ∼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 α-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.
-
isolation characterization and bioinformatic analysis of Calmodulin Binding protein cmbb reveals a novel tandem ip22 repeat common to many dictyostelium and mimivirus proteins
Biochemical and Biophysical Research Communications, 2006Co-Authors: Danton H Oday, Michael A Myre, Munmun Chatterjeechakraborty, Karsten Suhre, Sara E ChavezAbstract: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.
Michael A Myre - One of the best experts on this subject based on the ideXlab platform.
-
Calmodulin and Calmodulin Binding Proteins in Dictyostelium: A Primer.
International Journal of Molecular Sciences, 2020Co-Authors: Danton H. O'day, Ryan J. Taylor, Michael A MyreAbstract:Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium Binding and Calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-Binding proteins, the structure of Dictyostelium Calmodulin and the conformational changes effected by calcium ion Binding to its four EF hands are compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in Calmodulin Binding to target proteins are discussed with examples of the diversity of Calmodulin Binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize Calmodulin Binding proteins is covered followed by the ways Dictyostelium is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying Calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, Dictyostelium is in a position to become a leader in the field of Calmodulin research.
-
Calmodulin Binding Proteins and Alzheimer's Disease.
Journal of Alzheimer's Disease, 2015Co-Authors: Kristeen Eshak, Michael A MyreAbstract: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.
-
differentiation inducing factor 1 dif 1 induces gene and protein expression of the dictyostelium nuclear Calmodulin Binding protein nucleomorphin
Cellular Signalling, 2009Co-Authors: Danton H Oday, Yekaterina Poloz, Michael A MyreAbstract:Abstract 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.
-
isolation characterization and bioinformatic analysis of Calmodulin Binding protein cmbb reveals a novel tandem ip22 repeat common to many dictyostelium and mimivirus proteins
Biochemical and Biophysical Research Communications, 2006Co-Authors: Danton H Oday, Michael A Myre, Munmun Chatterjeechakraborty, Karsten Suhre, Sara E ChavezAbstract:Abstract 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 ∼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 α-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.
-
isolation characterization and bioinformatic analysis of Calmodulin Binding protein cmbb reveals a novel tandem ip22 repeat common to many dictyostelium and mimivirus proteins
Biochemical and Biophysical Research Communications, 2006Co-Authors: Danton H Oday, Michael A Myre, Munmun Chatterjeechakraborty, Karsten Suhre, Sara E ChavezAbstract: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.
Anthony Persechini - One of the best experts on this subject based on the ideXlab platform.
-
effects of combined phosphorylation at ser 617 and ser 1179 in endothelial nitric oxide synthase on ec50 ca2 values for Calmodulin Binding and enzyme activation
Journal of Biological Chemistry, 2009Co-Authors: Quangkim Tran, Jared Leonard, D J Black, Owen W Nadeau, Igor G Boulatnikov, Anthony PersechiniAbstract:We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC50(Ca2+) values for Calmodulin Binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC50(Ca2+) values for Calmodulin Binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca2+ concentration of 50–100 nm.
-
detection in living cells of ca2 dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a Calmodulin Binding sequence a new class of fluorescent indicators
Journal of Biological Chemistry, 1997Co-Authors: Valerie A Romoser, Patricia M Hinkle, Anthony PersechiniAbstract:We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the Calmodulin-Binding domain from smooth muscle myosin light chain kinase. When (Ca2+)4-Calmodulin is bound to the indicator (Kd = 0.4 nM), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases 6-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio-temporal changes in the fluorescence of the indicator. Changes in indicator fluorescence in these cells are coupled with no discernible lag (<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to approximately 1 microM. This observation suggests that the activity of a Calmodulin target with a typical 1 nM affinity for (Ca2+)4-Calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than Calmodulin.
-
detection in living cells of ca2 dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a Calmodulin Binding sequence a new class of fluorescent indicators
Journal of Biological Chemistry, 1997Co-Authors: Valerie A Romoser, Patricia M Hinkle, Anthony PersechiniAbstract:Abstract We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the Calmodulin-Binding domain from smooth muscle myosin light chain kinase. When (Ca2+)4-Calmodulin is bound to the indicator (K d = 0.4 nm), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases 6-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio-temporal changes in the fluorescence of the indicator. Changes in indicator fluorescence in these cells are coupled with no discernible lag (<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nm to ∼1 μm. This observation suggests that the activity of a Calmodulin target with a typical 1 nmaffinity for (Ca2+)4-Calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than Calmodulin.
Sara E Chavez - One of the best experts on this subject based on the ideXlab platform.
-
isolation characterization and bioinformatic analysis of Calmodulin Binding protein cmbb reveals a novel tandem ip22 repeat common to many dictyostelium and mimivirus proteins
Biochemical and Biophysical Research Communications, 2006Co-Authors: Danton H Oday, Michael A Myre, Munmun Chatterjeechakraborty, Karsten Suhre, Sara E ChavezAbstract:Abstract 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 ∼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 α-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.
-
isolation characterization and bioinformatic analysis of Calmodulin Binding protein cmbb reveals a novel tandem ip22 repeat common to many dictyostelium and mimivirus proteins
Biochemical and Biophysical Research Communications, 2006Co-Authors: Danton H Oday, Michael A Myre, Munmun Chatterjeechakraborty, Karsten Suhre, Sara E ChavezAbstract: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.
