Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance.

PubMed

Doherty, Colleen J; Van Buskirk, Heather A; Myers, Susan J; Thomashow, Michael F

2009-03-01

The Arabidopsis thaliana CBF cold response pathway plays a central role in cold acclimation. It is characterized by rapid cold induction of genes encoding the CBF1-3 transcription factors, followed by expression of the CBF gene regulon, which imparts freezing tolerance. Our goal was to further the understanding of the cis-acting elements and trans-acting factors involved in expression of CBF2. We identified seven conserved DNA motifs (CM), CM1 to 7, that are present in the promoters of CBF2 and another rapidly cold-induced gene encoding a transcription factor, ZAT12. The results presented indicate that in the CBF2 promoter, CM4 and CM6 have negative regulatory activity and that CM2 has both negative and positive activity. A Myc binding site in the CBF2 promoter was also found to have positive regulatory effects. Moreover, our results indicate that members of the calmodulin binding transcription activator (CAMTA) family of transcription factors bind to the CM2 motif, that CAMTA3 is a positive regulator of CBF2 expression, and that double camta1 camta3 mutant plants are impaired in freezing tolerance. These results establish a role for CAMTA proteins in cold acclimation and provide a possible point of integrating low-temperature calcium and calmodulin signaling with cold-regulated gene expression.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

PubMed

Heinen, R C; Diniz-Mendes, L; Silva, J T; Paschoalin, V M F

2006-11-01

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

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

NASA Technical Reports Server (NTRS)

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.

Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.

PubMed

Jung, Harin; Chung, Pil Joong; Park, Su-Hyun; Redillas, Mark Christian Felipe Reveche; Kim, Youn Shic; Suh, Joo-Won; Kim, Ju-Kon

2017-10-01

The AP2/ERF family is a plant-specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought-inducible OsERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI-1, -2, -3 and -4. A transactivation assay in yeast revealed that the C-terminal CMI-1 motif was essential for OsERF48 transcriptional activity. When OsERF48 was overexpressed in an either a root-specific (ROX O s ERF 48 ) or whole-body (OX O s ERF 48 ) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic (NT) controls. When plants were grown on a 40% polyethylene glycol-infused medium under in vitro drought conditions, ROX O s ERF 48 plants showed a more vigorous root growth than OX O s ERF 48 and NT plants. In addition, the ROX O s ERF 48 plants exhibited higher grain yield than OX O s ERF 48 and NT plants under field-drought conditions. We constructed a putative OsERF48 regulatory network by cross-referencing ROX O s ERF 48 root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in OsERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, OsCML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of OsERF48 by chromatin immunoprecipitation-qPCR analysis and a transient protoplast expression assay. Our results demonstrated that OsERF48 regulates OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Human Calmodulin Methyltransferase: Expression, Activity on Calmodulin, and Hsp90 Dependence

PubMed Central

Magen, Sophia; Magnani, Roberta; Haziza, Sitvanit; Hershkovitz, Eli; Houtz, Robert; Cambi, Franca; Parvari, Ruti

2012-01-01

Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34) has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels. We have identified 2 additional transcripts in cells of the 2p21 deletion syndrome patients that start from alternative exons positioned outside the deletion region. One of them starts in the 2nd known exon, the other in a novel exon. The transcript starting from the novel exon was also identified in a variety of tissues from normal individuals. These new transcripts are not expected to produce proteins. Immunofluorescent localization of tagged CaM KMT in HeLa cells indicates that it is present in both the cytoplasm and nucleus of cells whereas the short isoform is localized to the Golgi apparatus. Using Western blot analysis we show that the CaM KMT protein is broadly expressed in mouse tissues. Finally we demonstrate that the CaM KMT interacts with the middle portion of the Hsp90 molecular chaperon and is probably a client protein since it is degraded upon treatment of cells with the Hsp90 inhibitor geldanamycin. These findings suggest that the CaM KMT is the major, possibly the single, methyltransferase of calmodulin in human cells with a wide tissue distribution and is a novel Hsp90 client protein. Thus our data provides basic information for a gene potentially contributing to the patient phenotype of two contiguous gene deletion syndromes. PMID:23285036

Human calmodulin methyltransferase: expression, activity on calmodulin, and Hsp90 dependence.

PubMed

Magen, Sophia; Magnani, Roberta; Haziza, Sitvanit; Hershkovitz, Eli; Houtz, Robert; Cambi, Franca; Parvari, Ruti

2012-01-01

Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34) has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels. We have identified 2 additional transcripts in cells of the 2p21 deletion syndrome patients that start from alternative exons positioned outside the deletion region. One of them starts in the 2(nd) known exon, the other in a novel exon. The transcript starting from the novel exon was also identified in a variety of tissues from normal individuals. These new transcripts are not expected to produce proteins. Immunofluorescent localization of tagged CaM KMT in HeLa cells indicates that it is present in both the cytoplasm and nucleus of cells whereas the short isoform is localized to the Golgi apparatus. Using Western blot analysis we show that the CaM KMT protein is broadly expressed in mouse tissues. Finally we demonstrate that the CaM KMT interacts with the middle portion of the Hsp90 molecular chaperon and is probably a client protein since it is degraded upon treatment of cells with the Hsp90 inhibitor geldanamycin. These findings suggest that the CaM KMT is the major, possibly the single, methyltransferase of calmodulin in human cells with a wide tissue distribution and is a novel Hsp90 client protein. Thus our data provides basic information for a gene potentially contributing to the patient phenotype of two contiguous gene deletion syndromes.

Genes encoding calmodulin-binding proteins in the Arabidopsis genome

NASA Technical Reports Server (NTRS)

Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

2002-01-01

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

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

NASA Technical Reports Server (NTRS)

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.

Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici.

PubMed

Zheng, Zheng; Appiano, Michela; Pavan, Stefano; Bracuto, Valentina; Ricciardi, Luigi; Visser, Richard G F; Wolters, Anne-Marie A; Bai, Yuling

2016-01-01

The MLO (Mildew Locus O) gene family encodes plant-specific proteins containing seven transmembrane domains and likely acting in signal transduction in a calcium and calmodulin dependent manner. Some members of the MLO family are susceptibility factors toward fungi causing the powdery mildew disease. In tomato, for example, the loss-of-function of the MLO gene SlMLO1 leads to a particular form of powdery mildew resistance, called ol-2, which arrests almost completely fungal penetration. This type of penetration resistance is characterized by the apposition of papillae at the sites of plant-pathogen interaction. Other MLO homologs in Arabidopsis regulate root response to mechanical stimuli (AtMLO4 and AtMLO11) and pollen tube reception by the female gametophyte (AtMLO7). However, the role of most MLO genes remains unknown. In this work, we provide a genome-wide study of the tomato SlMLO gene family. Besides SlMLO1, other 15 SlMLO homologs were identified and characterized with respect to their structure, genomic organization, phylogenetic relationship, and expression profile. In addition, by analysis of transgenic plants, we demonstrated that simultaneous silencing of SlMLO1 and two of its closely related homologs, SlMLO5 and SlMLO8, confer higher level of resistance than the one associated with the ol-2 mutation. The outcome of this study provides evidence for functional redundancy among tomato homolog genes involved in powdery mildew susceptibility. Moreover, we developed a series of transgenic lines silenced for individual SlMLO homologs, which lay the foundation for further investigations aimed at assigning new biological functions to the MLO gene family.

Plant responses to environmental stress: regulation and functions of the Arabidopsis TCH genes

NASA Technical Reports Server (NTRS)

Braam, J.; Sistrunk, M. L.; Polisensky, D. H.; Xu, W.; Purugganan, M. M.; Antosiewicz, D. M.; Campbell, P.; Johnson, K. A.; McIntire, L. V. (Principal Investigator)

1997-01-01

Expression of the Arabidopsis TCH genes is markedly upregulated in response to a variety of environmental stimuli including the seemingly innocuous stimulus of touch. Understanding the mechanism(s) and factors that control TCH gene regulation will shed light on the signaling pathways that enable plants to respond to environmental conditions. The TCH proteins include calmodulin, calmodulin-related proteins and a xyloglucan endotransglycosylase. Expression analyses and localization of protein accumulation indicates that the potential sites of TCH protein function include expanding cells and tissues under mechanical strain. We hypothesize that at least a subset of the TCH proteins may collaborate in cell wall biogenesis.

Induced effect of Ca2+ on dalesconols A and B biosynthesis in the culture of Daldinia eschscholzii via calcium/calmodulin signaling.

PubMed

Lu, Yanhua; Pan, Zhenghua; Tao, Jun; An, Faliang

2018-02-01

Dalesconols (dalesconols A and B) were isolated from Daldinia eschscholzii and have remarkable immunosuppressive activity. In this study, the response of fungal growth, intra- and extracellular Ca 2+ , and dalesconols production after CaCl 2 addition were reported for the first time. After supplementation with 5 mM Ca 2+ at 24 h, dalesconols production reached 84.33 mg/L, which resulted in a 1.57-fold enhancement compared to the control. The key role of calcium/calmodulin signaling in dalesconols biosynthesis was confirmed by treatment with Ca 2+ channel and calmodulin inhibitors. The transcriptional levels of dalesconols biosynthetic genes were up-regulated after CaCl 2 addition and down-regulated after inhibitors were added. The results demonstrated that Ca 2+ addition induces dalesconols biosynthesis through up-regulation of dalesconols biosynthesis genes via regulation of calcium/calmodulin signaling. This study provided an efficient strategy for improving dalesconols production and would facilitate further research on the biosynthesis and regulation of dalesconols. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

López-Victorio, Carlos J.; Velez-delValle, Cristina; Beltrán-Langarica, Alicia

Highlights: ► EDF-1 participates early adipogenesis in 3T3F442A cells induced with Staurosporine/Dexamethasone. ► EDF-1 associates with CaM and Cn, most likely inactivating Cn. ► EDF-1/CaM complex seems to prevent NFATc1 activation by Cn. ► EDF-1 regulates the Cn/CaM/NFATc1 pathway during adipogenesis. ► EDF-1 may regulate the activation of Cn through a complex formation with CaM. - Abstract: The endothelial differentiation factor-1 (EDF-1) is a calmodulin binding protein that regulates calmodulin-dependent enzymes. In endothelial cells, this factor can form a protein complex with calmodulin. We analyzed the relationship between this factor and the members of calmodulin/calcineurin/nuclear factor of activated T-cells (NFAT)more » signaling pathway during adipogenesis of 3T3-F442A cells. We found that the expression of edf1 is upregulated during early adipogenesis, whereas that of calcineurin gene is lowered, suggesting that this pathway should be downregulated to allow for adipogenesis to occur. We also found that EDF-1 associates with calmodulin and calcineurin, most likely inactivating calcineurin. Our results showed that EDF-1 inactivates the calmodulin/calcineurin/NFAT pathway via sequestration of calmodulin, during early adipogenesis, and we propose a mechanism that negatively regulates the activation of calcineurin through a complex formation between EDF-1 and calmodulin. This finding raises the possibility that modulating this pathway might offer some alternatives to regulate adipose biology.« less

Genomic identification, phylogeny, and expression analysis of MLO genes involved in susceptibility to powdery mildew in Fragaria vesca.

PubMed

Miao, L X; Jiang, M; Zhang, Y C; Yang, X F; Zhang, H Q; Zhang, Z F; Wang, Y Z; Jiang, G H

2016-08-05

The MLO (powdery mildew locus O) gene family is important in resistance to powdery mildew (PM). In this study, all of the members of the MLO family were identified and analyzed in the strawberry (Fragaria vesca) genome. The strawberry contains at least 20 members of the MLO family, and the protein sequence contained between 171 and 1485 amino acids, with 0-34 introns. Chromosomal localization showed that the MLOs were unevenly distributed on each of the chromosomes, except for chromosome 4. The greatest number of MLOs (seven) was found on chromosome 3. A phylogenetic tree showed that the MLOs were divided into seven groups (I-VII), four of which consisted of MLOs from strawberry, Arabidopsis thaliana, rice, and maize, suggesting that these genes may have evolved after the divergence of monocots and dicots. Multiple sequence alignment showed that strawberry MLO candidates related to powdery mildew resistance possessed seven highly conserved transmembrane domains, a calmodulin-binding domain, and two conserved regions, all of which are important domains for powdery mildew resistance genes. Expressed sequence tag analysis revealed that the MLOs were induced by multiple abiotic stressors, including low and high temperature, drought, and high salinity. These findings will contribute to the functional characterization of MLOs related to PM susceptibility, and will assist in the development of disease resistance in strawberries.

Connecting RNA Processing to Abiotic Environmental Response in Arabidopsis: the role of a polyadenylation factor

NASA Astrophysics Data System (ADS)

Li, Q. Q.; Xu, R.; Hunt, A. G.; Falcone, D. L.

Plants are constantly challenged by numerous environmental stresses both biotic and abiotic It is clear that plants have evolved to counter these stresses using all but limited means We recently discovered the potential role of a messenger RNA processing factor namely the Arabidopsis cleavage and polyadenylation specificity factor 30 kDa subunit AtCPSF30 when a mutant deficient in this factor displayed altered responses to an array of abiotic stresses This AtCPSF30 mutant named oxt6 exhibited an elevated tolerance to oxidative stress Microarray experiments of oxt6 and its complemented lines revealed an altered gene expression profile among which were antioxidative defense genes Interestingly the same gene encoding AtCPSF30 can also be transcribed into a large transcript that codes for a potential splicing factor Both protein products have a domain for RNA binding and a calmodulin binding domain activities of which have been confirmed by biochemical assays Surprisingly binding of AtCPSF30 to calmodulin inhibits the RNA-binding activity of the protein Mutational analysis shows that a small part of the protein is responsible for calmodulin binding and point mutations in this region abolished both RNA binding activity and the inhibition of this activity by calmodulin Analyses of the potential splicing factor are on going and the results will be presented The interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through

Calmodulin Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Leishmania Identification and Typing

PubMed Central

Miranda, Aracelis; Samudio, Franklyn; González, Kadir; Saldaña, Azael; Brandão, Adeilton; Calzada, Jose E.

2016-01-01

A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy. PMID:27352873

Identification of Aspergillus sections Flavi, Nigri, and Fumigati and their differentiation using specific primers.

PubMed

Ashtiani, Nafiseh Mohebbi; Kachuei, Reza; Yalfani, Roozbeh; Harchegani, Asghar Beigi; Nosratabadi, Mohsen

2017-06-01

Aspergillus species are important in medicine, agriculture and various industries. The sections Fumigati, Flavi, and Nigri are the most important members of the Aspergillus genus. This study intended to identify and separate these three Aspergillus sections and to differentiate among them using specific primers. A bioinformatics study was initially performed to analyse the sequences of five genes, namely, beta-tubulin, calmodulin, the pre-rRNA processing protein Tsr1, the DNA-replication licensing factor Mcm7, and RNA polymerase II second largest subunit (RPB2) in the three Aspergillus sections using MEGA6 software and the NCBI database. Primers were designed to select genes for each of the Aspergillus sections being analysed. A total of 134 environmental and clinical Aspergillus species were isolated, purified and initially identified by colony morphology.. Subsequently, DNA was extracted using the phenol-chloroform method, specific primers were synthesized, PCR was performed for DNA from all isolates, and the results were compared to morphological characteristics. Of the 134 isolates tested, 56 were Nigri, 32 were Fumigati, 32 were Flavi, and the rest (14 isolates) belonged to other sections. The beta-tubulin and calmodulin genes were found to be the most suitable for differentiating among these three groups; the beta-tubulin gene was used for molecular identification of Aspergillus section Fumigati, and the calmodulin gene for identifying sections Flavi and Nigri.

MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type

PubMed Central

Wu, Hai; Naya, Francisco J.; McKinsey, Timothy A.; Mercer, Brian; Shelton, John M.; Chin, Eva R.; Simard, Alain R.; Michel, Robin N.; Bassel-Duby, Rhonda; Olson, Eric N.; Williams, R. Sanders

2000-01-01

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained. PMID:10790363

A population genetics analysis in clinical isolates of Sporothrix schenckii based on calmodulin and calcium/calmodulin-dependent kinase partial gene sequences.

PubMed

Rangel-Gamboa, Lucia; Martinez-Hernandez, Fernando; Maravilla, Pablo; Flisser, Ana

2018-02-02

Sporotrichosis is a subcutaneous mycosis that is caused by diverse species of Sporothrix. High levels of genetic diversity in Sporothrix isolates have been reported, but few population genetics analyses have been documented. To analyse the genetic variability and population genetics relations of Sporothrix schenckii Mexican clinical isolates and to compare them with other reported isolates. We studied the partial sequences of calmodulin and calcium/calmodulin-dependent kinase genes in 24 isolates; 22 from Mexico, one from Colombia, and one ATCC ® 6331™; the latter was used as a positive control. In total, 24 isolates were analysed. Phylogenetic, haplotype and population genetic analyses were performed with 24 sequences obtained by us and 345 sequences obtained from GenBank. The frequency of S. schenckii sensu stricto was 81% in the 22 Mexican isolates, while the remaining 19% were Sporothrix globosa. Mexican S. schenckii sensu stricto had high genetic diversity and was related to isolates from South America. In contrast, S. globosa showed one haplotype related to isolates from Asia, Brazil, Spain and the USA. In S. schenckii sensu stricto, S. brasiliensis and S. globosa, haplotype polymorphism (θ) values were higher than the nucleotide diversity data (π). In addition, Tajima's D plus Fu and Li's tests analyses displayed negative values, suggesting directional selection and arguing against the model of neutral evolution in these populations. In addition, analyses showed that calcium/calmodulin-dependent kinase was a suitable genetic marker to discriminate between common Sporothrix species. © 2018 Blackwell Verlag GmbH.

Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin'.

PubMed

Taniwaki, Marta Hiromi; Pitt, John I; Iamanaka, Beatriz T; Massi, Fernanda P; Fungaro, Maria Helena P; Frisvad, Jens C

2015-01-01

A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, β-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as bees and ants from the tree ecosystem in the Amazon rainforest. The species produces andrastin A, curvulic acid, penicillic acid and xanthoepocin, and has unique partial β-tubulin and calmodulin gene sequences. The holotype of P. excelsum is CCT 7772, while ITAL 7572 and IBT 31516 are cultures derived from the holotype.

Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin’

PubMed Central

Taniwaki, Marta Hiromi; Pitt, John I.; Iamanaka, Beatriz T.; Massi, Fernanda P.; Fungaro, Maria Helena P.; Frisvad, Jens C.

2015-01-01

A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, β-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as bees and ants from the tree ecosystem in the Amazon rainforest. The species produces andrastin A, curvulic acid, penicillic acid and xanthoepocin, and has unique partial β-tubulin and calmodulin gene sequences. The holotype of P. excelsum is CCT 7772, while ITAL 7572 and IBT 31516 are cultures derived from the holotype. PMID:26717519

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

PubMed Central

Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

2006-01-01

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein–protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 μM Ca2+, suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 μM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens. PMID:17003117

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

PubMed

Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

2006-10-03

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

Calmodulin binds to inv protein: implication for the regulation of inv function.

PubMed

Yasuhiko, Y; Imai, F; Ookubo, K; Takakuwa, Y; Shiokawa, K; Yokoyama, T

2001-12-01

Establishment of the left-right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left-right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca(2+) and is not observed in the presence of Ca(2+). Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two-cell stage randomized the left-right asymmetry of the embryo and altered the patterns of Xnr-1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left-right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left-right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium-calmodulin and discuss its implications.

Characterization of the Mantle Transcriptome of Yesso Scallop (Patinopecten yessoensis): Identification of Genes Potentially Involved in Biomineralization and Pigmentation

PubMed Central

Sun, Xiujun; Yang, Aiguo; Wu, Biao; Zhou, Liqing; Liu, Zhihong

2015-01-01

The Yesso scallop Patinopecten yessoensis is an economically important marine bivalve species in aquaculture and fishery in Asian countries. However, limited genomic resources are available for this scallop, which hampers investigations into molecular mechanisms underlying their unique biological characteristics, such as shell formation and pigmentation. Mantle is the special tissue of P. yessoensis that secretes biomineralization proteins inducing shell deposition as well as pigmentation on the shells. However, a current deficiency of transcriptome information limits insight into mechanisms of shell formation and pigmentation in this species. In this study, the transcriptome of the mantle of P. yessoensis was deeply sequenced and characterized using Illumina RNA-seq technology. A total of 86,521 unique transcripts are assembled from 55,884,122 reads that passed quality filters, and annotated, using Gene Ontology classification. A total of 259 pathways are identified in the mantle transcriptome, including the calcium signaling and melanogenesis pathways. A total of 237 unigenes that are homologous to 102 reported biomineralization genes are identified, and 121 unigenes that are homologous to 93 known proteins related to melanin biosynthesis are found. Twenty-three annotated unigenes, which are mainly homologous to calmodulin and related proteins, Ca2+/calmodulin-dependent protein kinase, adenylate/guanylate cyclase, and tyrosinase family are potentially involved in both biomineralization and melanin biosynthesis. It is suggested that these genes are probably not limited in function to induce shell deposition by calcium metabolism, but may also be involved in pigmentation of the shells of the scallop. This potentially supports the idea that there might be a link between calcium metabolism and melanin biosynthesis, which was previously found in vertebrates. The findings presented here will notably advance the understanding of the sophisticated processes of shell formation as well as shell pigmentation in P. yessoensis and other bivalve species, and also provide new evidence on gene expression for the understanding of pigmentation and biomineralization not only in invertebrates but also probably in vertebrates. PMID:25856556

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

PubMed Central

Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan

2013-01-01

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. PMID:24081981

WRKY transcription factors.

PubMed

Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

2010-05-01

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.

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

PubMed

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. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Inhibitors of the Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki

2007-11-23

Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP familymore » phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.« less

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

PubMed

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Day, Danton H., E-mail: danton.oday@utoronto.ca; Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario, Canada L5L 1C6; Huber, Robert J.

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 themore » 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.« less

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

PubMed

Michel, J B; Feron, O; Sase, K; Prabhakar, P; Michel, T

1997-10-10

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Urea-induced denaturation of human calcium/calmodulin-dependent protein kinase IV: a combined spectroscopic and MD simulation studies.

PubMed

Naz, Huma; Shahbaaz, Mohd; Haque, Md Anzarul; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2017-02-01

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme which belongs to the Ser/Thr kinase family. CaMKIV plays important role in varieties of biological processes such as gene expression regulation, memory consolidation, bone growth, T-cell maturation, sperm motility, regulation of microtubule dynamics, cell-cycle progression, and apoptosis. To measure stability parameters, urea-induced denaturation of CaMKIV was carried out at pH 7.4 and 25°C, using three different probes, namely far-UV CD, near-UV absorption, and tryptophan fluorescence. A coincidence of normalized denaturation curves of these optical properties suggests that urea-induced denaturation is a two-state process. Analysis of these denaturation curves gave values of 4.20 ± 0.12 kcal mol -1 , 2.95 ± 0.15 M, and 1.42 ± 0.06 kcal mol -1  M -1 for [Formula: see text] (Gibbs free energy change (ΔG D ) in the absence of urea), C m (molar urea concentration ([urea]) at the midpoint of the denaturation curve), and m (=∂ΔG D /∂[urea]), respectively. All these experimental observations have been fully supported by 30 ns molecular dynamics simulation studies.

Generic concepts in Nectriaceae

PubMed Central

Lombard, L.; van der Merwe, N.A.; Groenewald, J.Z.; Crous, P.W.

2015-01-01

The ascomycete family Nectriaceae (Hypocreales) includes numerous important plant and human pathogens, as well as several species used extensively in industrial and commercial applications as biodegraders and biocontrol agents. Members of the family are unified by phenotypic characters such as uniloculate ascomata that are yellow, orange-red to purple, and with phialidic asexual morphs. The generic concepts in Nectriaceae are poorly defined, since DNA sequence data have not been available for many of these genera. To address this issue we performed a multi-gene phylogenetic analysis using partial sequences for the 28S large subunit (LSU) nrDNA, the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), the large subunit of the ATP citrate lyase (acl1), the RNA polymerase II largest subunit (rpb1), RNA polymerase II second largest subunit (rpb2), α-actin (act), β-tubulin (tub2), calmodulin (cmdA), histone H3 (his3), and translation elongation factor 1-alpha (tef1) gene regions for available type and authentic strains representing known genera in Nectriaceae, including several genera for which no sequence data were previously available. Supported by morphological observations, the data resolved 47 genera in the Nectriaceae. We re-evaluated the status of several genera, which resulted in the introduction of six new genera to accommodate species that were initially classified based solely on morphological characters. Several generic names are proposed for synonymy based on the abolishment of dual nomenclature. Additionally, a new family is introduced for two genera that were previously accommodated in the Nectriaceae. PMID:26955195

Distinct Calcium Signaling Pathways Regulate Calmodulin Gene Expression in Tobacco1

PubMed Central

van der Luit, Arnold H.; Olivari, Claudio; Haley, Ann; Knight, Marc R.; Trewavas, Anthony J.

1999-01-01

Cold shock and wind stimuli initiate Ca2+ transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca2+ pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca2+ transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca2+ signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca2+ dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca2+ signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm. PMID:10557218

Voltage Dependence of a Neuromodulator-Activated Ionic Current.

PubMed

Gray, Michael; Golowasch, Jorge

2016-01-01

The neuromodulatory inward current (IMI) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that IMI loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of IMI is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMI voltage dependence in normal Ca(2+), but that, in conditions of low Ca(2+), calmodulin activators do not restore IMI voltage dependence. Further, we show evidence that CaMKII alters IMI voltage dependence. These results suggest that calmodulin is necessary but not sufficient for IMI voltage dependence. We therefore hypothesize that the Ca(2+)/calmodulin requirement for IMI voltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in IMI voltage dependence by a calmodulin inhibitor is due to CaSR endocytosis. Supporting this, preincubation with an endocytosis inhibitor prevented W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride)-induced loss of IMI voltage dependence, and a CaSR antagonist reduced IMI voltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating IMI voltage dependence. Finally, a Gβγ-subunit inhibitor also affects IMI voltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR.

Voltage Dependence of a Neuromodulator-Activated Ionic Current123

PubMed Central

2016-01-01

Abstract The neuromodulatory inward current (IMI) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that IMI loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of IMI is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMI voltage dependence in normal Ca2+, but that, in conditions of low Ca2+, calmodulin activators do not restore IMI voltage dependence. Further, we show evidence that CaMKII alters IMI voltage dependence. These results suggest that calmodulin is necessary but not sufficient for IMI voltage dependence. We therefore hypothesize that the Ca2+/calmodulin requirement for IMI voltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in IMI voltage dependence by a calmodulin inhibitor is due to CaSR endocytosis. Supporting this, preincubation with an endocytosis inhibitor prevented W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride)-induced loss of IMI voltage dependence, and a CaSR antagonist reduced IMI voltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating IMI voltage dependence. Finally, a Gβγ-subunit inhibitor also affects IMI voltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR. PMID:27257619

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways.

PubMed Central

Argüello-Astorga, G R; Herrera-Estrella, L R

1996-01-01

Regulation of plant gene transcription by light is mediated by multipartite cis-regulatory units. Previous attempts to identify structural features that are common to all light-responsive elements (LREs) have been unsuccessful. To address the question of what is needed to confer photoresponsiveness to a promoter, the upstream sequences from more than 110 light-regulated plant genes were analyzed by a new, phylogenetic-structural method. As a result, 30 distinct conserved DNA module arrays (CMAs) associated with light-responsive promoter regions were identified. Several of these CMAs have remained invariant throughout the evolutionary radiation of angiosperms and are conserved between homologous genes as well as between members of different gene families. The identified CMAs share a gene superfamily-specific core that correlates with the particular phytochrome-dependent transduction pathway that controls their expression, i.e. ACCTA(A/C)C(A/C) for the cGMP-dependent phenylpropanoid metabolism-associated genes, and GATA(A/T)GR for the Ca2+/calmodulin-dependent photosynthesis-associated nuclear genes. In addition to suggesting a general model for the functional and structural organization of LREs, the data obtained in this study indicate that angiosperm LREs probably evolved from complex cis-acting elements involved in regulatory processes other than photoregulation in gymnosperms. PMID:8938415

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

NASA Technical Reports Server (NTRS)

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.

Proteomic Analysis of Prostate Cancer Field Effect

DTIC Science & Technology

2011-02-01

Homo sapiens] profilin 1 [Homo sapiens] enolase 1 [Homo sapiens] sorbitol dehydrogenase [Homo sapiens] calmodulin 1 [Homo sapiens] apolipoprotein... sorbitol dehydrogenase [Homo sapiens] solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 [Homo sapiens] DJ-1

Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: calmodulin dendrograms show significant lack of parallelism

NASA Technical Reports Server (NTRS)

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.

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

NASA Technical Reports Server (NTRS)

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.

Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Tam, Emily W T; Chen, Jonathan H K; Lau, Eunice C L; Ngan, Antonio H Y; Fung, Kitty S C; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

2014-04-01

Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: Characterization by Internal Transcribed Spacer, β-Tubulin, and Calmodulin Gene Sequencing, Metabolic Fingerprinting, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Tam, Emily W. T.; Chen, Jonathan H. K.; Lau, Eunice C. L.; Ngan, Antonio H. Y.; Fung, Kitty S. C.; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung

2014-01-01

Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability. PMID:24452174

Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1(BmLsd)

USDA-ARS?s Scientific Manuscript database

The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

Regulation of RYR1 activity by Ca(2+) and calmodulin

NASA Technical Reports Server (NTRS)

Rodney, G. G.; Williams, B. Y.; Strasburg, G. M.; Beckingham, K.; Hamilton, S. L.

2000-01-01

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.

Sex-Dependent Up-Regulation of Two Splicing Factors, Psf and Srp20, during Hippocampal Memory Formation

ERIC Educational Resources Information Center

Antunes-Martins, Ana; Mizuno, Keiko; Irvine, Elaine E.; Lepicard, Eve M.; Giese, K. Peter

2007-01-01

Gene transcription is required for long-term memory (LTM) formation. LTM formation is impaired in a male-specific manner in mice lacking either of the two Ca[superscript 2+] / calmodulin-dependent kinase kinase ("Camkk") genes. Since altered transcription was suggested to cause these impairments in LTM formation, we used microarrays to screen for…

Static and kinetic studies of calmodulin and melittin complex.

PubMed

Itakura, M; Iio, T

1992-08-01

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.

Ca2+-dependent desensitization of TRPV2 channels is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate.

PubMed

Mercado, Jose; Gordon-Shaag, Ariela; Zagotta, William N; Gordon, Sharona E

2010-10-06

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca(2+)-dependent desensitization, similar to the other TRPVs, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or on coexpression of mutant calmodulin but was sensitive to changes in membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), suggesting a role of membrane PIP(2) in TRPV2 desensitization. Simultaneous confocal imaging and electrophysiological recording of cells expressing TRPV2 and a fluorescent PIP(2)-binding probe demonstrated that TRPV2 desensitization was concomitant with depletion of PIP(2). We conclude that the decrease in PIP(2) levels on channel activation underlies a major component of Ca(2+)-dependent desensitization of TRPV2 and may play a similar role in other TRP channels.

Ca2+-dependent Desensitization of TRPV2 Channels is Mediated by Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate

PubMed Central

Mercado, Jose; Gordon-Shaag, Ariela; Zagotta, William N.; Gordon, Sharona E.

2010-01-01

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca2+-dependent desensitization, similar to the other TRPV’s, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV’s channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or upon coexpression of mutant calmodulin but was sensitive to changes in membrane phosphatidylinositol (4,5)-bisphosphate (PIP2) suggesting a role of membrane PIP2 in TRPV2 desensitization. Simultaneous confocal imaging and electrophysiological recording of cells expressing TRPV2 and a fluorescent PIP2-binding probe demonstrated that TRPV2 desensitization was concomitant with depletion of PIP2. We conclude that the decrease in PIP2 levels upon channel activation underlies a major component of Ca2+-dependent desensitization of TRPV2 and may play a similar role in other TRP channels. PMID:20926660

Seawater Acidification and Elevated Temperature Affect Gene Expression Patterns of the Pearl Oyster Pinctada fucata

PubMed Central

Liu, Wenguang; Huang, Xiande; Lin, Jianshi; He, Maoxian

2012-01-01

Oceanic uptake of anthropogenic carbon dioxide results in decrease in seawater pH and increase in temperature. In this study, we demonstrated the synergistic effects of elevated seawater temperature and declined seawater pH on gene expression patterns of aspein, calmodulin, nacrein, she-7-F10 and hsp70 in the pearl oyster Pinctada fucata. Under ‘business-as-usual’ scenarios, four treatments were examined: (1) ambient pH (8.10) and ambient temperature (27°C) (control condition), (2) ambient pH and elevated temperature (+3°C), (3) declined pH (7.70) and ambient temperature, (4) declined pH and elevated temperature. The results showed that under warming and acidic seawater conditions, expression of aspein and calmodulin showed no significant differences among different time point in condition 8.10 T. But the levels of aspein and calmodulin in conditions 8.10 T+3, 7.70 T and 7.70 T+3, and levels of nacrein, she-7-F10 in all the four treatments changed significantly. Low pH and pH×temperature interaction influenced the expression of aspein and calmodulin significantly after hours 48 and 96. Significant effects of low pH and pH×temperature interaction on the expression of nacrein were observed at hour 96. The expression level of she-7-F10 was affected significantly by pH after hours 48 and 96. The expression of hsp70 was significantly affected by temperature, pH, temperature×pH interaction at hour 6, and by temperature×pH interaction at hour 24. This study suggested that declined pH and pH×temperature interaction induced down regulation of calcification related genes, and the interaction between declined seawater pH and elevated temperature caused up regulation of hsp70 in P. facata. These results demonstrate that the declined seawater pH and elevated temperature will impact the physiological process, and potentially the adaptability of P. fucata to future warming and acidified ocean. PMID:22438983

NM23 proteins: innocent bystanders or local energy boosters for CFTR?

PubMed

Muimo, Richmond; Alothaid, Hani Mm; Mehta, Anil

2018-03-01

NM23 proteins NDPK-A and -B bind to the cystic fibrosis (CF) protein CFTR in different ways from kinases such as PKA, CK2 and AMPK or linkers to cell calcium such as calmodulin and annexins. NDPK-A (not -B) interacts with CFTR through reciprocal AMPK binding/control, whereas NDPK-B (not -A) binds directly to CFTR. NDPK-B can activate G proteins without ligand-receptor coupling, so perhaps NDPK-B's binding influences energy supply local to a nucleotide-binding site (NBD1) needed for CFTR to function. Curiously, CFTR (ABC-C7) is a member of the ATP-binding cassette (ABC) protein family that does not obey 'clan rules'; CFTR channels anions and is not a pump, regulates disparate processes, is itself regulated by multiple means and is so pleiotropic that it acts as a hub that orchestrates calcium signaling through its consorts such as calmodulin/annexins. Furthermore, its multiple partners make CFTR dance to different tunes in different cellular and subcellular locations as it recycles from the plasma membrane to endosomes. CFTR function in airway apical membranes is inhibited by smoking which has been dubbed 'acquired CF'. CFTR alone among family members possesses a trap for other proteins that it unfurls as a 'fish-net' and which bears consensus phosphorylation sites for many protein kinases, with PKA being the most canonical. Recently, the site of CFTR's commonest mutation has been proposed as a knock-in mutant that alters allosteric control of kinase CK2 by log orders of activity towards calmodulin and other substrates after CFTR fragmentation. This link from CK2 to calmodulin that binds the R region invokes molecular paths that control lumen formation, which is incomplete in the tracheas of some CF-affected babies. Thus, we are poised to understand the many roles of NDPK-A and -B in CFTR function and, especially lumen formation, which is defective in the gut and lungs of many CF babies.

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

NASA Technical Reports Server (NTRS)

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.

Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

PubMed Central

Schmalzigaug, Robert; Ye, Qunrui; Berchtold, Martin W

2001-01-01

Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII−/− cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII−/− cells. Despite higher Ca2+ resting levels, the CaMII−/− cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes. PMID:11454062

Molecular cloning and expression of the calmodulin gene from guinea pig hearts.

PubMed

Feng, Rui; Liu, Yan; Sun, Xuefei; Wang, Yan; Hu, Huiyuan; Guo, Feng; Zhao, Jinsheng; Hao, Liying

2015-06-01

The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases.

Antibodies to calmodulin during experimental Trypanosoma brucei rhodesiense infections in rabbits.

PubMed Central

Ruben, L; Patton, C L

1985-01-01

Calmodulin is an intracellular Ca2+ receptor protein which regulates a wide variety of enzymatic processes in eukaryotic cells examined in detail. Native calmodulin is not antigenic in rabbits because of its small size, high degree of amino acid sequence conservation and hydrophobicity. African trypanosomes contain a novel calmodulin which is structurally distinct from bovine brain and Tetrahymena calmodulins. In the present study, we examine the antibody response towards these calmodulins during chronic Trypanosoma brucei rhodesiense infections. Injection of purified trypanosome calmodulin into rabbits stimulates the production of specific IgG antibodies which recognize trypanosome, but not bovine brain or Tetrahymena calmodulins. By contrast, during chronic T. brucei infections in rabbits, antibodies (IgG + IgM + IgA) that recognize trypanosome, Tetrahymena and mammalian calmodulins arise. When only IgG antibodies are evaluated from infection sera, the major response is against mammalian and Tetrahymena calmodulins. Significantly fewer IgG antibodies are measured in the infection sera which recognize trypanosome calmodulin, while the non-specific control protein, chicken ovalbumin, is not recognized. Peak IgG antibody responses against calmodulin occur between Days 30-34 post-infection. Competition assays indicate that Tetrahymena and mammalian calmodulins are recognized at identical epitopes which are distinct from epitopes on trypanosome calmodulin. We conclude that, in the context of chronic T. brucei infections in rabbits, antibodies arise which are able to recognize mammalian host calmodulin. Images Figure 1 PMID:2414212

Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses

PubMed Central

Yue, Runqing; Lu, Caixia; Sun, Tao; Peng, Tingting; Han, Xiaohua; Qi, Jianshuang; Yan, Shufeng; Tie, Shuanggui

2015-01-01

The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the −1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca2+ signaling in maize tolerance to environmental stresses. PMID:26284092

Galpha13 regulates MEF2-dependent gene transcription in endothelial cells: role in angiogenesis.

PubMed

Liu, Guoquan; Han, Jingyan; Profirovic, Jasmina; Strekalova, Elena; Voyno-Yasenetskaya, Tatyana A

2009-01-01

The alpha subunit of heterotrimeric G13 protein is required for the embryonic angiogenesis (Offermanns et al., Science 275:533-536, 1997). However, the molecular mechanism of Galpha13-dependent angiogenesis is not understood. Here, we show that myocyte-specific enhancer factor-2 (MEF2) mediates Galpha13-dependent angiogenesis. Our data showed that constitutively activated Galpha13Q226L stimulated MEF2-dependent gene transcription. In addition, downregulation of endogenous Galpha13 inhibited thrombin-stimulated MEF2-dependent gene transcription in endothelial cells. Both Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and histone deacetylase 5 (HDAC5) were involved in Galpha13-mediated MEF2-dependent gene transcription. Galpha13Q226L also increased Ca(2+)/calmodulin-independent CaMKIV activity, while dominant negative mutant of CaMKIV inhibited MEF2-dependent gene transcription induced by Galpha13Q226L. Furthermore, Galpha13Q226L was able to derepress HDAC5-mediated repression of gene transcription and induce the translocation of HDAC5 from nucleus to cytoplasm. Finally, downregulation of endogenous Galpha13 and MEF2 proteins in endothelial cells reduced cell proliferation and capillary tube formation. Decrease of endothelial cell proliferation that was caused by the Galpha13 downregulation was partially restored by the constitutively active MEF2-VP16. Our studies suggest that MEF2 proteins are an important component in Galpha13-mediated angiogenesis.

Decoding Ca2+ signals in plants

NASA Technical Reports Server (NTRS)

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

2004-01-01

Different input signals create their own characteristic Ca2+ fingerprints. These fingerprints are distinguished by frequency, amplitude, duration, and number of Ca2+ oscillations. Ca(2+)-binding proteins and protein kinases decode these complex Ca2+ fingerprints through conformational coupling and covalent modifications of proteins. This decoding of signals can lead to a physiological response with or without changes in gene expression. In plants, Ca(2+)-dependent protein kinases and Ca2+/calmodulin-dependent protein kinases are involved in decoding Ca2+ signals into phosphorylation signals. This review summarizes the elements of conformational coupling and molecular mechanisms of regulation of the two groups of protein kinases by Ca2+ and Ca2+/calmodulin in plants.

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

PubMed

Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

2004-09-10

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

Altered binding of /sup 125/I-labeled calmodulin to a 46. 5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tallant, E.A.; Wallace, R.W.

1987-02-01

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 Ca/sup 2 +//calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblastsmore » or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of /sup 125/I-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.« less

The Potassium Channel, Kir3.4 Participates in Angiotensin II-Stimulated Aldosterone Production by a Human Adrenocortical Cell Line

PubMed Central

Oki, Kenji; Plonczynski, Maria W.; Lam, Milay Luis; Gomez-Sanchez, Elise P.

2012-01-01

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4. PMID:22798349

NRIP is newly identified as a Z-disc protein, activating calmodulin signaling for skeletal muscle contraction and regeneration.

PubMed

Chen, Hsin-Hsiung; Chen, Wen-Pin; Yan, Wan-Lun; Huang, Yuan-Chun; Chang, Szu-Wei; Fu, Wen-Mei; Su, Ming-Jai; Yu, I-Shing; Tsai, Tzung-Chieh; Yan, Yu-Ting; Tsao, Yeou-Ping; Chen, Show-Li

2015-11-15

Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity. © 2015. Published by The Company of Biologists Ltd.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria.

PubMed

Chauhan, Arjun; Sharma, J N; Modgil, Manju; Siddappa, Sundaresha

2018-05-29

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus. Copyright © 2018 Elsevier B.V. All rights reserved.

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

PubMed Central

Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

2011-01-01

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

PubMed Central

1992-01-01

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1469047

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

PubMed

Heo, W D; Lee, S H; Kim, M C; Kim, J C; Chung, W S; Chun, H J; Lee, K J; Park, C Y; Park, H C; Choi, J Y; Cho, M J

1999-01-19

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, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass

DOE PAGES

Frazier, Taylor P.; Palmer, Nathan A.; Xie, Fuliang; ...

2016-11-08

Switchgrass ( Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass. In this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain,more » jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from ‘Alamo’, a rust-resistant switchgrass cultivar, and ‘Dacotah’, a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar ‘Summer’ plants indicated that the expression of some of these RGHs was developmentally regulated. Our results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop.« less

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frazier, Taylor P.; Palmer, Nathan A.; Xie, Fuliang

Switchgrass ( Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass. In this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain,more » jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from ‘Alamo’, a rust-resistant switchgrass cultivar, and ‘Dacotah’, a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar ‘Summer’ plants indicated that the expression of some of these RGHs was developmentally regulated. Our results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop.« less

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

PubMed Central

Li, Changlin; Cai, Xiangyu; Sun, Haili; Bai, Ting; Zheng, Xilong; Zhou, Xing Wang; Chen, Xiongwen; Gill, Donald L.; Li, Jing; Tang, Xiang D.

2011-01-01

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are 3 δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target. PMID:21554860

New insights into the operative network of FaEO, an enone oxidoreductase from Fragaria x ananassa Duch.

PubMed

Collu, Gabriella; Farci, Domenica; Esposito, Francesca; Pintus, Francesca; Kirkpatrick, Joanna; Piano, Dario

2017-05-01

The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.

Enzymatic assay for calmodulins based on plant NAD kinase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 formore » 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.« less

Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants.

PubMed

Hu, Xiuli; Jiang, Mingyi; Zhang, Jianhua; Zhang, Aying; Lin, Fan; Tan, Mingpu

2007-01-01

* Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Treatment with ABA or H(2)O(2) led to significant increases in the concentration of cytosolic Ca(2+) in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 (CaM1) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists. * Pretreatments with CaM antagonists almost completely inhibited ABA-induced H(2)O(2) production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA. * Our results suggest that Ca(2+)-CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca(2+)-CaM and H(2)O(2) plays a pivotal role in ABA signaling.

Overexpression of TaCML20, a calmodulin-like gene, enhances water soluble carbohydrate accumulation and yield in wheat.

PubMed

Kalaipandian, Sundaravelpandian; Xue, Gang-Ping; Rae, Anne L; Glassop, Donna; Bonnett, Graham D; McIntyre, Lynne C

2018-06-14

Calcium (Ca 2+ ) is a universal messenger that mediates intracellular responses to extracellular stimuli in living organisms. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are the important Ca 2+ sensors in plants that decode Ca 2+ -signatures to execute downstream intracellular level responses. Several studies indicate the interlinking of Ca 2+ and sugar signalling in plants, however, no genes have been functionally characterized to provide molecular evidence. Our study found that expression of TaCML20 was significantly correlated with water soluble carbohydrate (WSC) concentrations in recombinant inbred lines in wheat. TaCML20 has four EF-hand motifs that may facilitate the binding of Ca 2+ . To explore the role of CML20, we generated TaCML20 overexpressing transgenic lines in wheat. These lines accumulated higher WSC concentrations in the shoots, and we also found a significantly increased transcript level of sucrose:sucrose-1-fructosyltransferase (1-SST) in the internodes compared with the control plants. In addition, TaCML20 overexpressing plants showed significantly increased tillers per plant and also increased about 19% of grain weight per plant compared with control plants. The results also suggested a role for TaCML20 in drought stress, as its transcripts significantly increased in the shoots of wild-type plants under water deficit. These results uncovered the role of CML20 in determining multiple traits in wheat. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Analysis of the state of posttranslational calmodulin methylation in developing pea plants. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Sukheung; Roberts, D.M.

1990-07-01

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of ({sup 3}H)methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated andmore » green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.« less

Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

PubMed Central

Park, Miyoung; Martins, Vicente P.; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

2011-01-01

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis. PMID:21437209

Myosin light chains: Teaching old dogs new tricks

PubMed Central

Heissler, Sarah M; Sellers, James R

2014-01-01

The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amoebae into flagellates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.H.; Walsh, C.J.

1988-06-01

The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins ..cap alpha..- and BETA-tubulin and flagellar calmodulin, as well as a coordinately regulated poly(A)/sup +/ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)/sup +/ RNAs common to ameobae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in ameobae increased 3- to 10-fold. The observedmore » changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flagellar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. gruberi amebae to form visible flagella.« less

Fas Binding to Calmodulin Regulates Apoptosis in Osteoclasts*

PubMed Central

Wu, Xiaojun; Ahn, Eun-Young; McKenna, Margaret A.; Yeo, Hyeonju; McDonald, Jay M.

2005-01-01

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca2+ receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536–4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 μm, and trifluoperazine, 10 μm, induce 7.3 ± 1.8-fold and 5.3 ± 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661–5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 μm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr−cg mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr−cg mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr−cg mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 ± 0.2-fold in Lpr−cg-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis. PMID:15965236

The Calmodulin-related Calcium Sensor CML42 Plays a Role in Trichome Branching*

PubMed Central

Dobney, Stephanie; Chiasson, David; Lam, Polly; Smith, Steven P.; Snedden, Wayne A.

2009-01-01

Calcium (Ca2+) is a key second messenger in eukaryotes where it regulates a diverse array of cellular processes in response to external stimuli. An important Ca2+ sensor in both animals and plants is calmodulin (CaM). In addition to evolutionarily conserved CaM, plants possess a unique family of CaM-like (CML) proteins. The majority of these CMLs have not yet been studied, and investigation into their physical properties and cellular functions will provide insight into Ca2+ signal transduction in plants. Here we describe the characterization of CML42, a 191-amino acid Ca2+-binding protein from Arabidopsis. Ca2+ binding to recombinant CML42 was assessed by fluorescence spectroscopy, NMR spectroscopy, microcalorimetry, and CD spectroscopy. CML42 displays significant α-helical secondary structure, binds three molecules of Ca2+ with affinities ranging from 30 to 430 nm, and undergoes a Ca2+-induced conformational change that results in the exposure of one or more hydrophobic regions. Gene expression analysis revealed CML42 transcripts at various stages of development and in many cell types, including the support cells, which surround trichomes (leaf hairs) on the leaf surface. Using yeast two-hybrid screening we identified a putative CML42 interactor; kinesin-interacting Ca2+-binding protein (KIC). Because KIC is a protein known to function in trichome development, we examined transgenic CML42 knockout plants and found that they possess aberrant trichomes with increased branching. Collectively, our data support a role for CML42 as a Ca2+ sensor that functions during cell branching in trichomes. PMID:19720824

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

NASA Technical Reports Server (NTRS)

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.

Correlation between calmodulin activity and gravitropic sensitivity in primary roots of maize

NASA Technical Reports Server (NTRS)

Stinemetz, C. L.; Kuzmanoff, K. M.; Evans, M. L.; Jarrett, H. W.

1987-01-01

Recent evidence indicates a role for calcium and calmodulin in the gravitropic response of primary roots of maize (Zea mays, L.). We examined this possibility by testing the relationship between calmodulin activity and gravitropic sensitivity in roots of the maize cultivars Merit and B73 x Missouri 17. Roots of the Merit cultivar require light to the gravitropically competent. The gravitropic response of the Missouri cultivar is independent of light. The occurrence of calmodulin in primary roots of these maize cultivars was tested by affinity gel chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with bovine brain calmodulin as standard. The distribution of calmodulin activity was measured using both the phosphodiesterase and NAD kinase assays for calmodulin. These assays were performed on whole tissue segments, crude extracts, and purified extracts. In light-grown seedlings of the Merit cultivar or in either dark- or light-grown seedlings of the Missouri cultivar, calmodulin activity per millimeter of root tissue was about 4-fold higher in the apical millimeter than in the subtending 3 millimeters. Calmodulin activity was very low in the apical millimeter of roots of dark-grown (gravitropically nonresponsive) seedlings of the Merit cultivar. Upon illumination, the calmodulin activity in the apical millimeter increased to a level comparable to that of light-grown seedlings and the roots became gravitropically competent. The time course of the development of gravitropic sensitivity following illumination paralleled the time course of the increase in calmodulin activity in the apical millimeter of the root. The results are consistent with the suggestion that calmodulin plays an important role in the gravitropic response of roots.

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

NASA Technical Reports Server (NTRS)

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.

Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification

PubMed Central

Kinzer-Ursem, Tamara L.

2018-01-01

As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this “next-generation” calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics. PMID:29864125

Knockdown of PLC-gamma-2 and calmodulin 1 genes sensitizes human cervical adenocarcinoma cells to doxorubicin and paclitaxel.

PubMed

Stanislaus, Anthony; Bakhtiar, Athirah; Salleh, Diyana; Tiash, Snigdha; Fatemian, Tahereh; Hossain, Sharif; Akaike, Toshihiro; Chowdhury, Ezharul Hoque

2012-06-18

RNA interference (RNAi) is a powerful approach in functional genomics to selectively silence messenger mRNA (mRNA) expression and can be employed to rapidly develop potential novel drugs against a complex disease like cancer. However, naked siRNA being anionic is unable to cross the anionic cell membrane through passive diffusion and therefore, delivery of siRNA remains a major hurdle to overcome before the potential of siRNA technology can fully be exploited in cancer. pH-sensitive carbonate apatite has recently been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis. Moreover, internalized siRNA was found to escape from the endosomes in a time-dependent manner and efficiently silence gene expression. Here we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes has led to the sensitization of a human cervical cancer cell line to doxorubicin- and paclitaxel depending on the dosage of the individual drug whereas no such enhancement in cell death was observed with cisplatin irrespective of the dosage following intracellular delivery of the siRNAs. Thus, PLCG2 and CALM1 genes are two potential targets for gene knockdown in doxorubicin and paclitaxel-based chemotherapy of cervical cancer.

A Genomic Response to Trace Fear Conditioning in the Amygdala of Female Rats After Developmental Exposure to Manganese

EPA Science Inventory

Increases in brain-derived neurotrophic factor (Bdnf), Ca2+/calmodulin-dependent protein kinase II alpha (Camk2a), and cyclic adenosine monophosphate (cAMP) response element binding (Creb1) gene expression have been associated with learning in a variety of different rodent studie...

Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

PubMed

de Souza, Tatiana de Arruda Campos Brasil; Graça-de Souza, Viviane Krominski; Lancheros, César Armando Contreras; Monteiro-Góes, Viviane; Krieger, Marco Aurélio; Goldenberg, Samuel; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2011-03-01

In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.

Long-term memory deficits in Pavlovian fear conditioning in Ca2+/calmodulin kinase kinase alpha-deficient mice.

PubMed

Blaeser, Frank; Sanders, Matthew J; Truong, Nga; Ko, Shanelle; Wu, Long Jun; Wozniak, David F; Fanselow, Michael S; Zhuo, Min; Chatila, Talal A

2006-12-01

Signaling by the Ca(2+)/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase alpha (CaMKKalpha) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKalpha mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context fear during both conditioning and long-term follow-up testing. They also exhibited impaired activation of the downstream kinase CaMKIV/Gr and its substrate, the transcription factor cyclic AMP-responsive element binding protein (CREB) upon fear conditioning. Unlike CaMKIV/Gr-deficient mice, the CaMKKalpha mutants exhibited normal long-term potentiation and normal levels of anxiety-like behavior. These results demonstrate a selective role for CaMKKalpha in contextual fear memory and suggest that different combinations of upstream and downstream components of the CaMK cascade may serve distinct physiological functions.

Multiple calmodulin-like proteins in Arabidopsis are induced by insect-derived (Spodoptera littoralis) oral secretion

PubMed Central

Vadassery, Jyothilakshmi; Scholz, Sandra S.; Mithöfer, Axel

2012-01-01

In plant cells, diverse environmental changes often induce transient elevation in the intracellular calcium concentrations, which are involved in signaling pathways leading to the respective cellular reactions. Therefore, these calcium elevations need to be deciphered into specific downstream responses. Calmodulin-like-proteins (CMLs) are calcium-sensing proteins present only in higher plants. They are involved in signaling processes induced by both abiotic as well as biotic stress factors. However, the role of CMLs in the interaction of plants with herbivorous insects is almost unknown. Here we show that in Arabidopsis thaliana a number of CMLs genes (CML9, 11,12,16,17 and 23) are upregulated due to treatments with oral secretion of larvae of the herbivorous insect Spodoptera littoralis. We identified that these genes belong to two groups that respond with different kinetics to the treatment with oral secretion. Our data indicate that signaling networks involving multiple CMLs very likely have important functions in plant defense against insect herbivores, in addition to their involvement in many other stress-induced processes in plants. PMID:22902684

Cross-Linking Proteins To Show Complex Formation: A Laboratory That Visually Demonstrates Calmodulin Binding to Calmodulin Kinase II.

ERIC Educational Resources Information Center

Porta, Angela R.

2003-01-01

Presents a laboratory experiment demonstrating the binding of calcium/calmodulin to calmodulin kinase II, which is important in the metabolic and physiological activities of the cell. Uses SDS polyacrylamide gel electrophoresis (PAGE). (YDS)

Apocalmodulin and Ca2+ calmodulin bind to the same region on the skeletal muscle Ca2+ release channel

NASA Technical Reports Server (NTRS)

Moore, C. P.; Rodney, G.; Zhang, J. Z.; Santacruz-Toloza, L.; Strasburg, G.; Hamilton, S. L.

1999-01-01

The skeletal muscle Ca2+ release channel (RYR1) is regulated by calmodulin in both its Ca2+-free (apocalmodulin) and Ca2+-bound (Ca2+ calmodulin) states. Apocalmodulin is an activator of the channel, and Ca2+ calmodulin is an inhibitor of the channel. Both apocalmodulin and Ca2+ calmodulin binding sites on RYR1 are destroyed by a mild tryptic digestion of the sarcoplasmic reticulum membranes, but calmodulin (either form), bound to RYR1 prior to tryptic digestion, protects both the apocalmodulin and Ca2+ calmodulin sites from tryptic destruction. The protected sites are after arginines 3630 and 3637 on RYR1. These studies suggest that both Ca2+ calmodulin and apocalmodulin bind to the same or overlapping regions on RYR1 and block access of trypsin to sites at amino acids 3630 and 3637. This sequence is part of a predicted Ca2+ CaM binding site of amino acids 3614-3642 [Takeshima, H., et al. (1989) Nature 339, 439-445].

Calcium/calmodulin and cAMP/protein kinase-A pathways regulate sperm motility in the stallion.

PubMed

Lasko, Jodi; Schlingmann, Karen; Klocke, Ann; Mengel, Grace Ann; Turner, Regina

2012-06-01

In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II β were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acrosome and flagellar principal piece. Additionally, inhibition of either calmodulin or protein kinase-A significantly reduced sperm motility without affecting viability. Following inhibition of calmodulin, motility was not restored with agonists of the cyclic AMP/protein kinase-A pathway. These data suggest that calcium/calmodulin and cyclic AMP/protein kinase-A pathways are involved in the regulation of stallion sperm motility. The failure of cyclic AMP/protein kinase-A agonists to restore motility of calmodulin inhibited sperm suggests that both pathways may be required to support normal motility. Copyright © 2012 Elsevier B.V. All rights reserved.

Rat leucine-rich protein binds and activates the promoter of the beta isoform of Ca2+/calmodulin-dependent protein kinase II gene.

PubMed

Ochiai, Nagahiro; Masumoto, Shuji; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Yamauchi, Takashi

2007-05-01

We previously found the neuronal cell-type specific promoter and binding partner of the beta isoform of Ca(2+)/calmodulin-dependent protein kinase II (beta CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of beta isoform of Ca(2+)/calmodulin-dependent protein kinase II of rat brain. Mol. Brain Res. 94, 35-47]. In the present study, we purified a protein that binds specifically a promoter region of beta CaM kinase II gene from a nuclear extract of the rat cerebellum using DEAE-cellulose column chromatography, ammonium sulfate fractionation, gel filtration and polyacrylamide gel electrophoresis. The purified protein was identified as rat leucine-rich protein 157 (rLRP157) using tandem mass spectrometry. Then, we prepared its cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly(A)(+)RNA of rat cerebellum. The rLRP157 cDNA was introduced into mouse neuroblastomaxrat glioma hybrid NG108-15 cells, and cells stably expressing rLRP157 (NG/LRP cells) were isolated. Binding of rLRP157 with the promoter sequence was confirmed by electrophoretic mobility shift assay using nuclear extract of NG/LRP cells. A luciferase reporter gene containing a promoter of beta CaM kinase II was transiently expressed in NG/LRP cells. Under the conditions, the promoter activity was enhanced about 2.6-fold in NG/LRP cells as compared with wild-type cells. The expression of rLRP157 mRNA was paralleled with that of beta CaM kinase II in the adult and embryo rat brain detected by in situ hybridization. Nuclear localization of rLRP157 was confirmed using GFP-rLRP157 fusion protein investigated under a confocal microscope. These results indicate that rLRP157 is one of the proteins binding to, and regulating the activity of, the promoter of beta CaM kinase II.

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

NASA Technical Reports Server (NTRS)

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.

FKBP12.6 protects heart from AngII-induced hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways in vivo and in vitro.

PubMed

Xiao, Yun-Fei; Zeng, Zhi-Xiong; Guan, Xiao-Hui; Wang, Ling-Fang; Wang, Chan-Juan; Shi, Huidong; Shou, Weinian; Deng, Ke-Yu; Xin, Hong-Bo

2018-04-22

We previously observed that disruption of FK506-binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)-induced cardiac hypertrophy in mice, whereas the adenovirus-mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)-induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6 -/- ) mice and cardiac-specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini-pump. The results showed that FKBP12.6 deficiency aggravated AngII-induced cardiac hypertrophy, while cardiac-specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII-induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII-induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca 2+ ([Ca 2+ ]i), in which the protein significantly inhibited the key Ca 2+ /calmodulin-dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF-2, AKT/Glycogen synthase kinase 3β (GSK3β)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII-induced cardiac hypertrophy through inhibiting Ca 2+ /calmodulin-mediated signalling pathways. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess1[C][W][OA

PubMed Central

González, Alberto; Cabrera, M. de los Ángeles; Henríquez, M. Josefa; Contreras, Rodrigo A.; Morales, Bernardo; Moenne, Alejandra

2012-01-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H2O2) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H2O2, ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H2O2 increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H2O2 accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H2O2. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H2O2, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases. PMID:22234999

Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium-dependent protein kinases in Ulva compressa exposed to copper excess.

PubMed

González, Alberto; Cabrera, M de Los Ángeles; Henríquez, M Josefa; Contreras, Rodrigo A; Morales, Bernardo; Moenne, Alejandra

2012-03-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H(2)O(2), ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H(2)O(2) increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H(2)O(2) accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H(2)O(2). In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H(2)O(2), and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.

Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective.

PubMed

La Verde, Valentina; Dominici, Paola; Astegno, Alessandra

2018-04-30

Ca 2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca 2+ -binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca 2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca 2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca 2+ sensors is also presented.

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.

PubMed

Montgomery, H J; Romanov, V; Guillemette, J G

2000-02-18

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.

Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.

PubMed

Romero, I; Maldonado, A M; Eraso, P

1997-03-15

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of approximately 1 microM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

Invasive Sino-Orbital Mycosis in an Aplastic Anemia Patient Caused by Neosartorya laciniosa

PubMed Central

Malejczyk, Kathy; Sigler, Lynne; Gibas, Connie Fe C.

2013-01-01

We report the first case of Neosartorya laciniosa invasive sinusitis involving the orbit in an immunocompromised male with aplastic anemia. Treatment included surgical debridement with enucleation of the eye and combination voriconazole and micafungin therapy followed by voriconazole alone. The fungus was identified using sequencing of partial benA and calmodulin genes. PMID:23345294

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

PubMed

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

2008-05-01

Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide (H(2)O(2)), calcium (Ca(2+))-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, H(2)O(2), and CaCl(2) 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 Ca(2+) inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca(2+)-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 Ca(2+) in protoplasts of mesophyll cells and in the expression of calmodulin 1 (CaM1) 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 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca(2+) inhibitors and CaM antagonists. Our results suggest that Ca(2+)-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H(2)O(2)-induced antioxidant defense in leaves of maize plants.

Calcium and calmodulin are involved in blue light induction of the gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas.

PubMed Central

Im, C S; Matters, G L; Beale, S I

1996-01-01

The Chlamydomonas reinhardtii nuclear gene gsa, which encodes the early chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase (GSAT), is specifically induced by blue light in cells synchronized in a 12-hr-light and 12-hr-dark regime. Light induction required the presence of a nitrogen source in the incubation medium. Maximal induction also required acetate. However, in the absence of acetate, partial induction occurred when Ca2+ was present in the medium at concentrations of > or = 1 microM. The Ca2+ channel-blocking agents Nd3+ and nifedipine partially inhibited the external Ca(2+)-supported induction of GSAT mRNA but did not inhibit acetate-supported induction. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited both external Ca(2+)-supported and acetate-supported induction. The Ca2+ ionophore A23187 caused a transient induction in the dark. These results suggest that Ca2+ and calmodulin are involved in the signal transduction pathway linking blue light perception to the induction of GSAT mRNA. The electron transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited acetate-supported induction of GSAT mRNA but did not inhibit external Ca(2+)-supported induction. It is proposed that in the presence of acetate, an internal pool of Ca2+ can be mobilized as a second message, whereas in the absence of acetate, internal Ca2+ is not available but the requirement for Ca2+ can be partially met by an external Ca2+ source. The mobilization of internal Ca2+ may require energy derived from metabolism of acetate. PMID:8989881

Phylogeny of Plant CAMTAs and Role of AtCAMTAs in Nonhost Resistance to Xanthomonas oryzae pv. oryzae

PubMed Central

Rahman, Hafizur; Yang, Juan; Xu, You-Ping; Munyampundu, Jean-Pierre; Cai, Xin-Zhong

2016-01-01

Calmodulin-binding transcription activator (CAMTA) constitutes one of the most important Ca2+/CaM-regulated transcription factor families in plants. Nevertheless, the phylogeny, protein interaction network, and role in nonhost resistance of plant CAMTAs are not well understood. In this study, 200 CAMTA genes were identified from 35 species representing four major plant lineages. The CAMTA genes were conserved in multicellular land plants but absent in unicellular eukaryotes, and were likely to emerge from the fusion of two separate genes encoding a CAMTA-like protein and an IQ/CaM binding motif containing protein, respectively, in the embryophyta lineage ancestor. Approximately one fourth of plant CAMTAs did not contain a TIG domain. This non-TIG class of CAMTAs seems to have newly evolved through mutation of some key amino acids in the TIG domain of flowering land plants after divergence from the non-flowering plants. Phylogenetic analysis classified CAMTA proteins into three major groups and nine distinct subgroups, a result supported by protein domain and motif conservation analyses. Most (59.0 and 21.5%) of the identified CAMTA genes contained 12 or 11 introns, respectively. Gene duplication, intron invasion, enlargement and turnover, as well as exon rearrangements and skipping have apparently occurred during evolution of the CAMTA family. Moreover, 38 potential interactors of six Arabidopsis CAMTAs were predicted and 10 predicted target genes of AtCAMTA3 exhibited changes in expression between Atcamta3 mutants and wild-type plants. The majority of predicted interactors are transcription factors and/or Ca2+/CaM-regulated proteins, suggesting that transcriptional regulation of the target genes might be the dominant functional mechanism of AtCAMTAs, and AtCAMTAs might act together with other Ca2+ signaling components to regulate Ca2+-related biological processes. Furthermore, functional analyses employing Atcamta mutants revealed that AtCAMTA3 negatively regulated the immunity triggered by flg22 and nonhost resistance to Xanthomonas oryzae pv. oryzae via repressing accumulation of reactive oxygen species probably by targeting CBP60G, EDS1, and NDR1 and involving SA pathway. PMID:26973658

Phylogeny of Plant CAMTAs and Role of AtCAMTAs in Nonhost Resistance to Xanthomonas oryzae pv. oryzae.

PubMed

Rahman, Hafizur; Yang, Juan; Xu, You-Ping; Munyampundu, Jean-Pierre; Cai, Xin-Zhong

2016-01-01

Calmodulin-binding transcription activator (CAMTA) constitutes one of the most important Ca(2+)/CaM-regulated transcription factor families in plants. Nevertheless, the phylogeny, protein interaction network, and role in nonhost resistance of plant CAMTAs are not well understood. In this study, 200 CAMTA genes were identified from 35 species representing four major plant lineages. The CAMTA genes were conserved in multicellular land plants but absent in unicellular eukaryotes, and were likely to emerge from the fusion of two separate genes encoding a CAMTA-like protein and an IQ/CaM binding motif containing protein, respectively, in the embryophyta lineage ancestor. Approximately one fourth of plant CAMTAs did not contain a TIG domain. This non-TIG class of CAMTAs seems to have newly evolved through mutation of some key amino acids in the TIG domain of flowering land plants after divergence from the non-flowering plants. Phylogenetic analysis classified CAMTA proteins into three major groups and nine distinct subgroups, a result supported by protein domain and motif conservation analyses. Most (59.0 and 21.5%) of the identified CAMTA genes contained 12 or 11 introns, respectively. Gene duplication, intron invasion, enlargement and turnover, as well as exon rearrangements and skipping have apparently occurred during evolution of the CAMTA family. Moreover, 38 potential interactors of six Arabidopsis CAMTAs were predicted and 10 predicted target genes of AtCAMTA3 exhibited changes in expression between Atcamta3 mutants and wild-type plants. The majority of predicted interactors are transcription factors and/or Ca(2+)/CaM-regulated proteins, suggesting that transcriptional regulation of the target genes might be the dominant functional mechanism of AtCAMTAs, and AtCAMTAs might act together with other Ca(2+) signaling components to regulate Ca(2+)-related biological processes. Furthermore, functional analyses employing Atcamta mutants revealed that AtCAMTA3 negatively regulated the immunity triggered by flg22 and nonhost resistance to Xanthomonas oryzae pv. oryzae via repressing accumulation of reactive oxygen species probably by targeting CBP60G, EDS1, and NDR1 and involving SA pathway.

A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium

PubMed Central

2012-01-01

The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription. PMID:22273182

Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea.

PubMed

Yamashita, Masashi; Sueyoshi, Noriyuki; Yamada, Hiroki; Katayama, Syouichi; Senga, Yukako; Takenaka, Yasuhiro; Ishida, Atsuhiko; Kameshita, Isamu; Shigeri, Yasushi

2018-04-20

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca 2+ /CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca 2+ /CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca 2+ -signaling in C. cinerea.

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

PubMed

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

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

PubMed

Sixt, S U; Jennissen, H P; Winterhalter, M; Laub, M

2010-10-25

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 proteasome 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.

Comparative Analysis of Anther Transcriptome Profiles of Two Different Rice Male Sterile Lines Genotypes under Cold Stress.

PubMed

Bai, Bin; Wu, Jun; Sheng, Wen-Tao; Zhou, Bo; Zhou, Li-Jie; Zhuang, Wen; Yao, Dong-Ping; Deng, Qi-Yun

2015-05-18

Rice is highly sensitive to cold stress during reproductive developmental stages, and little is known about the mechanisms of cold responses in rice anther. Using the HiSeq™ 2000 sequencing platform, the anther transcriptome of photo thermo sensitive genic male sterile lines (PTGMS) rice Y58S and P64S (Pei'ai64S) were analyzed at the fertility sensitive stage under cold stress. Approximately 243 million clean reads were obtained from four libraries and aligned against the oryza indica genome and 1497 and 5652 differentially expressed genes (DEGs) were identified in P64S and Y58S, respectively. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for these DEGs. Functional classification of DEGs was also carried out. The DEGs common to both genotypes were mainly involved in signal transduction, metabolism, transport, and transcriptional regulation. Most of the DEGs were unique for each comparison group. We observed that there were more differentially expressed MYB (Myeloblastosis) and zinc finger family transcription factors and signal transduction components such as calmodulin/calcium dependent protein kinases in the Y58S comparison group. It was also found that ribosome-related DEGs may play key roles in cold stress signal transduction. These results presented here would be particularly useful for further studies on investigating the molecular mechanisms of rice responses to cold stress.

Comparative Analysis of Anther Transcriptome Profiles of Two Different Rice Male Sterile Lines Genotypes under Cold Stress

PubMed Central

Bai, Bin; Wu, Jun; Sheng, Wen-Tao; Zhou, Bo; Zhou, Li-Jie; Zhuang, Wen; Yao, Dong-Ping; Deng, Qi-Yun

2015-01-01

Rice is highly sensitive to cold stress during reproductive developmental stages, and little is known about the mechanisms of cold responses in rice anther. Using the HiSeq™ 2000 sequencing platform, the anther transcriptome of photo thermo sensitive genic male sterile lines (PTGMS) rice Y58S and P64S (Pei’ai64S) were analyzed at the fertility sensitive stage under cold stress. Approximately 243 million clean reads were obtained from four libraries and aligned against the oryza indica genome and 1497 and 5652 differentially expressed genes (DEGs) were identified in P64S and Y58S, respectively. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for these DEGs. Functional classification of DEGs was also carried out. The DEGs common to both genotypes were mainly involved in signal transduction, metabolism, transport, and transcriptional regulation. Most of the DEGs were unique for each comparison group. We observed that there were more differentially expressed MYB (Myeloblastosis) and zinc finger family transcription factors and signal transduction components such as calmodulin/calcium dependent protein kinases in the Y58S comparison group. It was also found that ribosome-related DEGs may play key roles in cold stress signal transduction. These results presented here would be particularly useful for further studies on investigating the molecular mechanisms of rice responses to cold stress. PMID:25993302

The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions

PubMed Central

Xu, Weihui; Meng, Yan; Surana, Priyanka; Fuerst, Greg; Nettleton, Dan; Wise, Roger P.

2015-01-01

Plants have evolved complex regulatory mechanisms to control a multi-layered defense response to microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSINs, small knottin-like peptides in barley, wheat, and rice, are highly induced by attack from fungal pathogens, in particular, the obligate biotrophic fungus, Blumeria graminis f. sp. hordei (Bgh), causal agent of barley powdery mildew. Previous research indicated that Blufensin1 (Bln1) functions as a negative regulator of basal defense mechanisms. In the current report, we show that BLN1 and BLN2 can both be secreted to the apoplast and Barley stripe mosaic virus (BSMV)-mediated overexpression of Bln2 increases susceptibility of barley to Bgh. Bimolecular fluorescence complementation (BiFC) assays signify that BLN1 and BLN2 can interact with each other, and with calmodulin. We then used BSMV-induced gene silencing to knock down Bln1, followed by Barley1 GeneChip transcriptome analysis, to identify additional host genes influenced by Bln1. Analysis of differential expression revealed a gene set enriched for those encoding proteins annotated to nuclear import and the secretory pathway, particularly Importin α1-b and Sec61 γ subunits. Further functional analysis of these two affected genes showed that when silenced, they also reduced susceptibility to Bgh. Taken together, we postulate that Bln1 is co-opted by Bgh to facilitate transport of disease-related host proteins or effectors, influencing the establishment of Bgh compatibility on its barley host. PMID:26089830

Role of the MAGUK protein family in synapse formation and function.

PubMed

Oliva, Carlos; Escobedo, Pía; Astorga, César; Molina, Claudia; Sierralta, Jimena

2012-01-01

Synaptic function is crucially dependent on the spatial organization of the presynaptic and postsynaptic apparatuses and the juxtaposition of both membrane compartments. This precise arrangement is achieved by a protein network at the submembrane region of each cell that is built around scaffold proteins. The membrane-associated guanylate kinase (MAGUK) family of proteins is a widely expressed and well-conserved group of proteins that plays an essential role in the formation and regulation of this scaffolding. Here, we review general features of this protein family, focusing on the discs large and calcium/calmodulin-dependent serine protein kinase subfamilies of MAGUKs in the formation, function, and plasticity of synapses. Copyright © 2011 Wiley Periodicals, Inc.

Corticotropin-releasing hormone or dexamethasone regulates rat proopiomelanocortin transcription through Tpit/Pitx-responsive element in its promoter.

PubMed

Murakami, Itsuo; Takeuchi, Sakae; Kudo, Toshiyuki; Sutou, Shizuyo; Takahashi, Sumio

2007-05-01

Tpit/Pitx-responsive element (Tpit/PitxRE), which binds transcription factors Tpit and Pitx1, confers cell-type specific expression of proopiomelanocortin (POMC) gene in pituitary corticotrops where the gene expression is mainly regulated by corticotropin-releasing hormone (CRH) and glucocorticoids (Gcs). CRH stimulates POMC gene expression, which is mediated by the accumulation of intracellular cAMP and requires binding of Nur factors to Nur-responsive element (NurRE). Gcs antagonize NurRE-dependent POMC gene expression through direct interaction between glucocorticoid receptors and Nur factors. We examined whether Tpit/PitxRE and NurRE are involved in CRH/cAMP-induced activation and Gc-induced repression of POMC gene expression by reporter assay in AtT-20 corticotropic cells. Deletion and mutation of Tpit/PitxRE markedly reduced basal activity of the promoter, and those of NurRE decreased the levels of the CRH/cAMP-induced activation. Nifedipine, KN-62, and W-7, specific inhibitors of the L-type calcium channel, calmodulin-dependent protein kinase II, and calmodulin respectively, attenuated CRH/cAMP-induced activation of promoters with three copies of either Tpit/PitxRE or NurRE, indicating that both Tpit/PitxRE and NurRE mediate CRH-induced activation of POMC gene expression in a calcium-dependent manner. Deletion and mutation of Tpit/PitxRE abolished dexamethasone (DEX)-induced repression of POMC gene expression, while those of NurRE did not, indicating that Tpit/PitxRE predominantly mediates Gc-induced repression of POMC transcription. However, DEX treatment attenuated activities of promoters with three copies of either Tpit/PitxRE or NurRE, suggesting that Gcs act at NurRE as well as Tpit/PitxRE to repress POMC gene expression. We conclude that Tpit/PitxRE is an important element by which CRH and Gcs regulate the POMC gene expression.

The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

ERIC Educational Resources Information Center

Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

2016-01-01

The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

Association of Genetic Variation in Calmodulin and Left Ventricular Mass in Full-Term Newborns

PubMed Central

Gorący, Iwona; Gorący, Jarosław; Skonieczna-Żydecka, Karolina; Kaczmarczyk, Mariusz; Dawid, Grażyna; Ciechanowicz, Andrzej

2013-01-01

Calmodulin II (CALM2) gene polymorphism might be responsible for the variation in the left ventricular mass amongst healthy individuals. The aim was to evaluate the correlation between left ventricular mass (LVM) and g.474955027G>A (rs7565161) polymorphism adjacent to the CALM2 gene. Healthy Polish newborns (n = 206) were recruited. Two-dimensional M-mode echocardiography was used to assess LVM. Polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism and sequencing analyses. The carriers of the G allele of the CALM2 polymorphism had significantly higher left ventricular mass/weight (LVM/BW) values, when compared with newborns homozygous for the A allele (3.1 g/m2 versus 2.5 g/m2, P adjusted = 0.036). The AG genotype of CALM2 was associated with the highest values of LVM/BW, exhibiting a pattern of overdominance (2.9 g/kg versus 3.1 g/kg versus 2.5 g/kg, P adjusted = 0.037). The results of this study suggest that G>A CALM2 polymorphism may account for subtle variation in LVM at birth. PMID:24298550

Differential expression of calcium/calmodulin-regulated SlSRs in response to abiotic and biotic stresses in tomato fruit.

PubMed

Yang, Tianbao; Peng, Hui; Whitaker, Bruce D; Jurick, Wayne M

2013-07-01

Calcium has been shown to enhance stress tolerance, maintain firmness and reduce decay in fruits. Previously we reported that seven tomato SlSRs encode calcium/calmodulin-regulated proteins, and that their expressions are developmentally regulated during fruit development and ripening, and are also responsive to ethylene. To study their expressions in response to stresses encountered during postharvest handling, tomato fruit at the mature-green stage was subjected to chilling and wounding injuries, infected with Botrytis cinerea and treated with salicylic acid or methyl jasmonate. Gene expression studies revealed that the seven SlSRs differentially respond to different stress signals. SlSR2 was the only gene upregulated by all the treatments. SlSR4 acted as a late pathogen-induced gene; it was upregulated by salicylic acid and methyl jasmonate, but downregulated by cold treatment. SlSR3L was cold- and wound-responsive and was also induced by salicylic acid. SlSR1 and SlSR1L were repressed by cold, wounding and pathogen infection, but were upregulated by salicylic acid and methyl jasmonate. Overall, results of these expression studies indicate that individual SlSRs have distinct roles in responses to the specific stress signals, and SlSRs may act as a coordinator(s) connecting calcium-mediated signaling with other stress signal transduction pathways during fruit ripening and storage. © 2013 Scandinavian Plant Physiology Society.

Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

NASA Technical Reports Server (NTRS)

Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

1997-01-01

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

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

NASA Technical Reports Server (NTRS)

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.

Calmodulin point mutations affect Drosophila development and behavior.

PubMed

Nelson, H B; Heiman, R G; Bolduc, C; Kovalick, G E; Whitley, P; Stern, M; Beckingham, K

1997-12-01

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

Calmodulin Point Mutations Affect Drosophila Development and Behavior

PubMed Central

Nelson, H. B.; Heiman, R. G.; Bolduc, C.; Kovalick, G. E.; Whitley, P.; Stern, M.; Beckingham, K.

1997-01-01

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations. PMID:9409836

Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies.

PubMed

Naz, Huma; Shahbaaz, Mohd; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2016-06-01

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium-calmodulin dependent signal through a secondary messenger, Ca(2+), which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca(2+) concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0-11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0-4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV.

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

PubMed Central

Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

2012-01-01

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

FAK/Src family of kinases: protective or aggravating factor for ischemia reperfusion injury in nervous system?

PubMed

Bikis, Christos; Moris, Demetrios; Vasileiou, Ioanna; Patsouris, Eustratios; Theocharis, Stamatios

2015-04-01

The focal adhesion kinase (FAK) and the Src families of kinases are subfamilies of the non-receptor protein tyrosine kinases. FAK activity is regulated by gene amplification, alternative splicing and phosporylation/dephosphorylation. FAK/Src complex has been found to participate through various pathways in neuronal models of ischemia-reperfusion injury (IRI) with conflicting results. The aim of the present review is to summarize the currently available data on this subject. The MEDLINE/PubMed database was searched for publications with the medical subject heading IRI and FAK and/or Src, nervous system. We restricted our search till 2014. We identified 93 articles that were available in English as abstracts or/and full-text articles that were deemed appropriate for our review. FAK has been found to have a beneficial preconditioning effect on IRI through activation via the protein kinase C (PKC) pathway by anesthetic agents. Of great importance are the interactions between FAK/Src and VEGF that has been already detected as a protective mean for IRI. The effect of VEGF administration might depend on dose as well as on time of administration. A Ca(2+)/calmodulin-dependent protein kinase II or PKC inhibitors seem to have protective effects on IRI by inhibiting ion channels activation.

High-throughput transcriptome analysis of barley (Hordeum vulgare) exposed to excessive boron.

PubMed

Tombuloglu, Guzin; Tombuloglu, Huseyin; Sakcali, M Serdal; Unver, Turgay

2015-02-15

Boron (B) is an essential micronutrient for optimum plant growth. However, above certain threshold B is toxic and causes yield loss in agricultural lands. While a number of studies were conducted to understand B tolerance mechanism, a transcriptome-wide approach for B tolerant barley is performed here for the first time. A high-throughput RNA-Seq (cDNA) sequencing technology (Illumina) was used with barley (Hordeum vulgare), yielding 208 million clean reads. In total, 256,874 unigenes were generated and assigned to known peptide databases: Gene Ontology (GO) (99,043), Swiss-Prot (38,266), Clusters of Orthologous Groups (COG) (26,250), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (36,860), as determined by BLASTx search. According to the digital gene expression (DGE) analyses, 16% and 17% of the transcripts were found to be differentially regulated in root and leaf tissues, respectively. Most of them were involved in cell wall, stress response, membrane, protein kinase and transporter mechanisms. Some of the genes detected as highly expressed in root tissue are phospholipases, predicted divalent heavy-metal cation transporters, formin-like proteins and calmodulin/Ca(2+)-binding proteins. In addition, chitin-binding lectin precursor, ubiquitin carboxyl-terminal hydrolase, and serine/threonine-protein kinase AFC2 genes were indicated to be highly regulated in leaf tissue upon excess B treatment. Some pathways, such as the Ca(2+)-calmodulin system, are activated in response to B toxicity. The differential regulation of 10 transcripts was confirmed by qRT-PCR, revealing the tissue-specific responses against B toxicity and their putative function in B-tolerance mechanisms. Copyright © 2014. Published by Elsevier B.V.

Chronic ethanol increases calcium/calmodulin-dependent protein kinaseIIδ gene expression and decreases monoamine oxidase amount in rat heart muscles: Rescue effect of Zingiber officinale (ginger) extract.

PubMed

Heshmati, Elaheh; Shirpoor, Alireza; Kheradmand, Fatemeh; Alizadeh, Mohammad; Gharalari, Farzaneh Hosseini

2018-01-01

Association between chronic alcohol intake and cardiac abnormality is well known; however, the precise underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. This study investigated the effect of chronic ethanol exposure on calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) gene expression and monoamine oxidase (MAO) levels and histological changes in rat heart. It was also planned to find out whether Zingiber officinale (ginger) extract mitigated the abnormalities induced by ethanol in rat heart. Male wistar rats were divided into three groups of eight animals each: control, ethanol, and ginger extract treated-ethanol (GETE) groups. After 6 weeks of treatment, the results revealed a significant increase in CaMKIIδtotal and isoforms δ2 and δ3 of CaMKIIδ gene expression as well as a significant decrease in the MAO levels in the ethanol group compared to that in the control group. Moreover, compared to the control group, the ethanol group showed histological changes, such as fibrosis, heart muscle cells proliferation, myocyte hypertrophy, vacuolization, and focal lymphocytic infiltration. Consumption of ginger extract along with ethanol ameliorated CaMKIIδtotal. In addition, compared to the ethanol group, isoforms gene expression changed and increased the reduced MAO levels and mitigated heart structural changes. These findings indicate that ethanol-induced heart abnormalities may, in part, be associated with Ca 2+ homeostasis changes mediated by overexpression of CaMKIIδ gene and the decrease of MAO levels and that these effects can be alleviated by using ginger extract as an antioxidant and anti-inflammatory agent.

Gender-Specific Potential Inhibitory Role of Ca2+/Calmodulin Dependent Protein Kinase Phosphatase (CaMKP) in Pressure-Overloaded Mouse Heart

PubMed Central

Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

2014-01-01

Background Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Methodology and Principal Findings Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Conclusions/Significance Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases. PMID:24608696

Gender-specific potential inhibitory role of Ca2+/calmodulin dependent protein kinase phosphatase (CaMKP) in pressure-overloaded mouse heart.

PubMed

Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

2014-01-01

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.

Ca2+–calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

PubMed Central

Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

2011-01-01

Abstract Recent studies have demonstrated that changes in the activity of calcium–calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation–contraction (E–C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM–GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+–CaMKII–DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII. PMID:21486818

Calmodulin Lobes Facilitate Dimerization and Activation of Estrogen Receptor-α*

PubMed Central

Li, Zhigang; Zhang, Yonghong; Hedman, Andrew C.; Ames, James B.

2017-01-01

Estrogen receptor α (ER-α) is a nuclear hormone receptor that controls selected genes, thereby regulating proliferation and differentiation of target tissues, such as breast. Gene expression controlled by ER-α is modulated by Ca2+ via calmodulin (CaM). Here we present the NMR structure of Ca2+-CaM bound to two molecules of ER-α (residues 287–305). The two lobes of CaM bind to the same site on two separate ER-α molecules (residues 292, 296, 299, 302, and 303), which explains why CaM binds two molecules of ER-α in a 1:2 complex and stabilizes ER-α dimerization. Exposed glutamate residues in CaM (Glu-11, Glu-14, Glu-84, and Glu-87) form salt bridges with key lysine residues in ER-α (Lys-299, Lys-302, and Lys-303), which is likely to prevent ubiquitination at these sites and inhibit degradation of ER-α. Transfection of cells with full-length CaM slightly increased the ability of estrogen to enhance transcriptional activation by ER-α of endogenous estrogen-responsive genes. By contrast, expression of either the N- or C-lobe of CaM abrogated estrogen-stimulated transcription of the estrogen responsive genes pS2 and progesterone receptor. These data suggest that CaM-induced dimerization of ER-α is required for estrogen-stimulated transcriptional activation by the receptor. In light of the critical role of ER-α in breast carcinoma, our data suggest that small molecules that selectively disrupt the interaction of ER-α with CaM may be useful in the therapy of breast carcinoma. PMID:28174300

Inhibition of calcium/calmodulin kinase II alpha subunit expression results in epileptiform activity in cultured hippocampal neurons.

PubMed

Churn, S B; Sombati, S; Jakoi, E R; Severt, L; DeLorenzo, R J; Sievert, L

2000-05-09

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the alpha subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

Inhibition of calcium/calmodulin kinase II alpha subunit expression results in epileptiform activity in cultured hippocampal neurons

PubMed Central

Churn, Severn B.; Sombati, Sompong; Jakoi, Emma R.; Sievert, Lawrence; DeLorenzo, Robert J.

2000-01-01

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the α subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy. PMID:10779547

The Neuronal Calcium Sensor Protein Acrocalcin: A Potential Target of Calmodulin Regulation during Development in the Coral Acropora millepora

PubMed Central

Reyes-Bermudez, Alejandro; Miller, David J.; Sprungala, Susanne

2012-01-01

To understand the calcium-mediated signalling pathways underlying settlement and metamorphosis in the Scleractinian coral Acropora millepora, a predicted protein set derived from larval cDNAs was scanned for the presence of EF-hand domains (Pfam Id: PF00036). This approach led to the identification of a canonical calmodulin (AmCaM) protein and an uncharacterised member of the Neuronal Calcium Sensor (NCS) family of proteins known here as Acrocalcin (AmAC). While AmCaM transcripts were present throughout development, AmAC transcripts were not detected prior to gastrulation, after which relatively constant mRNA levels were detected until metamorphosis and settlement. The AmAC protein contains an internal CaM-binding site and was shown to interact in vitro with AmCaM. These results are consistent with the idea that AmAC is a target of AmCaM in vivo, suggesting that this interaction may regulate calcium-dependent processes during the development of Acropora millepora. PMID:23284743

Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

PubMed

Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

2017-11-01

We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

Reconstruction of calmodulin single-molecule FRET states, dye interactions, and CaMKII peptide binding by MultiNest and classic maximum entropy

NASA Astrophysics Data System (ADS)

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2013-08-01

We analyzed single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

Reconstruction of Calmodulin Single-Molecule FRET States, Dye-Interactions, and CaMKII Peptide Binding by MultiNest and Classic Maximum Entropy

PubMed Central

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2013-01-01

We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data. PMID:24223465

Reconstruction of Calmodulin Single-Molecule FRET States, Dye-Interactions, and CaMKII Peptide Binding by MultiNest and Classic Maximum Entropy.

PubMed

Devore, Matthew S; Gull, Stephen F; Johnson, Carey K

2013-08-30

We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca 2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

PubMed Central

Comte, M; Malnoë, A; Cox, J A

1986-01-01

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5. Images Fig. 2. PMID:3814096

Frequency of a natural truncated allele of MdMLO19 in the germplasm of Malus domestica.

PubMed

Pessina, Stefano; Palmieri, Luisa; Bianco, Luca; Gassmann, Jennifer; van de Weg, Eric; Visser, Richard G F; Magnago, Pierluigi; Schouten, Henk J; Bai, Yuling; Riccardo Velasco, R; Malnoy, Mickael

2017-01-01

Podosphaera leucotricha is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of MdMLO19 in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five MLO genes ( MdMLO5, MdMLO7, MdMLO11, MdMLO18 , and MdMLO19 ), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional mlo allele has been found.

A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Julie Anne Roden, Branids Belt, Jason Barzel Ross, Thomas Tachibana, Joe Vargas, Mary Beth Mudgett

2004-11-23

The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resultedmore » in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.« less

An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

NASA Technical Reports Server (NTRS)

Yang, T.; Poovaiah, B. W.

2000-01-01

35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

[Gene expression and activity regulation of two calmodulin binding protein kinases in tobacco seedling].

PubMed

Hua, Wei; Li, Rong-Jun; Liang, Shu-Ping; Lu, Ying-Tang

2005-06-01

Two different calmodulin-binding protein kinase cDNAs (NtCBK1/2) have been isolated from tobacco. To understand the CBK protein activity regulation, we compared the activity regulation of NtCBK1 and NtCBK2 by pH, Mg(2+) concentration and Na(+) concentration. We found the autophosphorylation of NtCBK1/2 reached the maximum in pH 7.5 and 8 respectively; Mg(2+) and Na(+) shown different effects on the activity of NtCBKs, high and low Mg(2+) concentrations both inhibited the activity of NtCBKs, but Na+ had little effect on the kinase activity. In addition, to obtain further insight about the physiological roles of individual NtCBKs, we detected the expression profiles of CBKs. The results revealed different patterns of expression of NtCBK1 and NtCBK2. Both are largely expressed in leaf and flower; but in stem and root, NtCBK1 gene had stronger expression than NtCBK2. NtCBK2 expression was induced by GA treatment, while NtCBK1 expression remained unchanged under GA treatment. Expression of both NtCBK1 and NtCBK2 increased in response to salt stress, the former to a greater extent, and both expressions did not change under high/low temperature, drought, NAA and ABA treatments.

Coping with Stresses: Roles of Calcium- and Calcium/Calmodulin-Regulated Gene Expression[W][OA

PubMed Central

Reddy, Anireddy S.N.; Ali, Gul S.; Celesnik, Helena; Day, Irene S.

2011-01-01

Abiotic and biotic stresses are major limiting factors of crop yields and cause billions of dollars of losses annually around the world. It is hoped that understanding at the molecular level how plants respond to adverse conditions and adapt to a changing environment will help in developing plants that can better cope with stresses. Acquisition of stress tolerance requires orchestration of a multitude of biochemical and physiological changes, and most of these depend on changes in gene expression. Research during the last two decades has established that different stresses cause signal-specific changes in cellular Ca2+ level, which functions as a messenger in modulating diverse physiological processes that are important for stress adaptation. In recent years, many Ca2+ and Ca2+/calmodulin (CaM) binding transcription factors (TFs) have been identified in plants. Functional analyses of some of these TFs indicate that they play key roles in stress signaling pathways. Here, we review recent progress in this area with emphasis on the roles of Ca2+- and Ca2+/CaM-regulated transcription in stress responses. We will discuss emerging paradigms in the field, highlight the areas that need further investigation, and present some promising novel high-throughput tools to address Ca2+-regulated transcriptional networks. PMID:21642548

Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

PubMed

Kano, Rui; Okubo, Miki; Siew, Han Hock; Kamata, Hiroshi; Hasegawa, Atsuhiko

2015-04-01

Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia. © 2015 Blackwell Verlag GmbH.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schallreuter, K.U.; Gibbons, N.C.J.; Zothner, C.

Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10{sup -3} M H{sub 2}O{sub 2}. 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{sup -3}M H{sub 2}O{sub 2} oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H{sub 2}O{sub 2} utilising {sup 45}calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activitiesmore » 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 H{sub 2}O{sub 2}-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo.« less

Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development.

PubMed

Al-Shanti, Nasser; Stewart, Claire E

2009-11-01

The loss of muscle mass with age and disuse has a significant impact on the physiological and social well-being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross-sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age-related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin-like growth factor I (IGF-I) and Ca(2+)/calmodulin-dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF-I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca(2+)/calmodulin-dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T-cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca(2+) sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca(2+)/calmodulin-dependent kinases pathways (via CaMKs) or calmodulin-dependent serine/threonine phosphatases (via calcineurin). (3) How calmodulin kinases alter transcription in the nucleus through the phosphorylation, deactivation and translocation of histone deacetylase 4 (HDAC4) from the nucleus to the cytoplasm. (4) How calcineurin transmits signals to the nucleus through the dephosphorylation and translocation of NFAT from the cytoplasm to the nucleus.

A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

NASA Technical Reports Server (NTRS)

Porter Moore, C.; Zhang, J. Z.; Hamilton, S. L.

1999-01-01

Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

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

NASA Technical Reports Server (NTRS)

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.

Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

PubMed Central

Bosser, R; Faura, M; Serratosa, J; Renau-Piqueras, J; Pruschy, M; Bachs, O

1995-01-01

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei. PMID:7823935

Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen.

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

AKAP150-mediated TRPV1 sensitization is disrupted by calcium/calmodulin

PubMed Central

2011-01-01

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. PMID:21569553

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

PubMed

Chaudhury, Sraboni; Bal, Manjot; Belugin, Sergei; Shapiro, Mark S; Jeske, Nathaniel A

2011-05-14

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. 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. the results from these studies indicate that calcium/calmodulin interferes with the association of AKAP150 with TRPV1, potentially extending resensitization of the channel.

Protein Kinases in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1999-09-01

studies identical at the amino acid level to calcium/calmodulin-dependent may provide insight into mechanisms of growth control and DNA protein kinase I...human homologues of these kinases(19, 20 ). Amino acid conservation in the coding region between mouse and human Hunk is greater than 90% identical. While...genes (13, 14). Over the past 4 years , several of the mRNA and protein levels (39-46). These findings clearly dem- these breast cancer susceptibility

Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane.

PubMed

Alli, Abdel A; Bao, Hui-Fang; Liu, Bing-Chen; Yu, Ling; Aldrugh, Summer; Montgomery, Darrice S; Ma, He-Ping; Eaton, Douglas C

2015-09-01

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane. Copyright © 2015 the American Physiological Society.

Plants, symbiosis and parasites: a calcium signalling connection.

PubMed

Harper, Jeffrey F; Harmon, Alice

2005-07-01

A unique family of protein kinases has evolved with regulatory domains containing sequences that are related to Ca(2+)-binding EF-hands. In this family, the archetypal Ca(2+)-dependent protein kinases (CDPKs) have been found in plants and some protists, including the malarial parasite, Plasmodium falciparum. Recent genetic evidence has revealed isoform-specific functions for a CDPK that is essential for Plasmodium berghei gametogenesis, and for a related chimeric Ca(2+) and calmodulin-dependent protein kinase (CCaMK) that is essential to the formation of symbiotic nitrogen-fixing nodules in plants. In Arabidopsis thaliana, the analysis of 42 isoforms of CDPK and related kinases is expected to delineate Ca(2+) signalling pathways in all aspects of plant biology.

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

PubMed

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 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-dependent signaling at the receptor level through multiple interaction sites. FRET biosensors represent a simple method to identify unknown calmodulin-binding domains in G protein-coupled receptors and to quantitatively assess binding properties.

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

PubMed Central

Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

2016-01-01

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin (CaM) Variants in Long QT Syndrome (LQTS) and Functional Characterization of a Novel LQTS-Associated CaM Missense Variant, E141G

PubMed Central

Calvert, Melissa L.; Tester, David J.; Kryshtal, Dmytro; Hwang, Hyun Seok; Johnson, Christopher N.; Chazin, Walter J.; Loporcaro, Christina G.; Shah, Maully; Papez, Andrew L.; Lau, Yung R.; Kanter, Ronald; Knollmann, Bjorn C.; Ackerman, Michael J.

2016-01-01

Background Calmodulin (CaM) is encoded by three genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca2+ and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All of these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results Thirty-nine genetically elusive LQTS cases underwent whole exome sequencing to identify CaM variants. Non-synonymous CaM variants were overrepresented significantly in this heretofore LQTS cohort (15.4%) compared to exome aggregation consortium (0.04%; p<0.0001). When the clinical sequelae of these 6 CaM-positive cases was compared to the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 8 months, an average QTc of 679 ms, and a high prevalence of cardiac arrest. Functional characterization of one novel variant, E141G-CaM, revealed an 11-fold reduction in Ca2+ binding affinity and a functionally-dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions Overall, 15% of our genetically elusive LQTS cohort harbored non-synonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history. PMID:26969752

CML24, Regulated in Expression by Diverse Stimuli, Encodes a Potential Ca2+ Sensor That Functions in Responses to Abscisic Acid, Daylength, and Ion Stress1

PubMed Central

Delk, Nikkí A.; Johnson, Keith A.; Chowdhury, Naweed I.; Braam, Janet

2005-01-01

Changes in intracellular calcium (Ca2+) levels serve to signal responses to diverse stimuli. Ca2+ signals are likely perceived through proteins that bind Ca2+, undergo conformation changes following Ca2+ binding, and interact with target proteins. The 50-member calmodulin-like (CML) Arabidopsis (Arabidopsis thaliana) family encodes proteins containing the predicted Ca2+-binding EF-hand motif. The functions of virtually all these proteins are unknown. CML24, also known as TCH2, shares over 40% amino acid sequence identity with calmodulin, has four EF hands, and undergoes Ca2+-dependent changes in hydrophobic interaction chromatography and migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs, and transcript levels are increased from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA), and indole-3-acetic acid. However, CML24 protein accumulation changes were not detectable. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress; in regions undergoing growth; in vascular tissues and various floral organs; and in stomata, trichomes, and hydathodes. CML24-underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, are defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4, and MgCl2. MgCl2 tolerance is not due to reduced uptake or to elevated Ca2+ accumulation. Together, these data present evidence that CML24, a gene expressed in diverse organs and responsive to diverse stimuli, encodes a potential Ca2+ sensor that may function to enable responses to ABA, daylength, and presence of various salts. PMID:16113225

Isolation, characterization and immunolocalization of a seed dominant CaM from finger millet (Eleusine coracana L. Gartn.) for studying its functional role in differential accumulation of calcium in developing grains.

PubMed

Kumar, Anil; Mirza, Neelofar; Charan, Tara; Sharma, Netrapal; Gaur, Vikram Singh

2014-03-01

To understand the exceptional high grain calcium accumulation in finger millet grains, a calmodulin (CaM) gene that is strongly expressed during developing spikes of high grain calcium genotype was further characterized. Using 5'-3' RACE, the full-length CaM open reading frame (ORF) was isolated and the deduced protein sequence showed the presence of four characteristic EF motifs. Phylogenetic analysis showed that the finger millet CaM (Eleusine coracana calmodulin [EcCaM]) was identical to the rice CaM 1-1. Southern hybridization showed the presence of at least four copies of CaM gene that might be located on different regions of the finger millet "AABB" genome. Immunodetection using monospecific polyclonal anti-EcCaM antibodies revealed that EcCaM is localized in the embryo and aleurone layer and accumulates in higher amounts in high grain calcium genotype compared to the low grain calcium genotype. Furthermore, in silico analysis showed that EcCaM interacts with aquaporin which indicates that calcium is probably delivered to developing spike via mass flow of water. These results indicate that higher expression of CaM might cause greater stimulation of the downstream calcium transport machinery operative in the aleurone layer leading to the higher calcium accumulation in the grains of high grain calcium genotype.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Microfluidic free-flow electrophoresis for the discovery and characterisation of calmodulin binding partners

NASA Astrophysics Data System (ADS)

Herling, Therese; Linse, Sara; Knowles, Tuomas

2015-03-01

Non-covalent and transient protein-ligand interactions are integral to cellular function and malfunction. Key steps in signalling and regulatory pathways rely on reversible non-covalent protein-protein binding or ion chelation. Here we present a microfluidic free-flow electrophoresis method for detecting and characterising protein-ligand interactions in solution. We apply this method to probe the binding equilibria of calmodulin, a central protein to calcium signalling pathways. In this study we characterise the specific binding of calmodulin to phosphorylase kinase, a known target, and creatine kinase, which we identify as a putative binding partner through a protein array screen and surface plasmon resonance experiments. We verify the interaction between calmodulin and creatine kinase in solution using free-flow electrophoresis and investigate the effect of calcium and sodium chloride on the calmodulin-ligand binding affinity in free solution without the presence of a potentially interfering surface. Our results demonstrate the general applicability of quantitative microfluidic electrophoresis to characterise binding equilibria between biomolecules in solution.

Synergy of cAMP and calcium signaling pathways in CFTR regulation

PubMed Central

Bozoky, Zoltan; Ahmadi, Saumel; Milman, Tal; Kim, Tae Hun; Du, Kai; Di Paola, Michelle; Pasyk, Stan; Pekhletski, Roman; Keller, Jacob P.; Bear, Christine E.; Forman-Kay, Julie D.

2017-01-01

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport. PMID:28242698

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

NASA Technical Reports Server (NTRS)

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.

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

NASA Technical Reports Server (NTRS)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

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

NASA Technical Reports Server (NTRS)

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.

CML8, an Arabidopsis Calmodulin-Like Protein, Plays a Role in Pseudomonas syringae Plant Immunity.

PubMed

Zhu, Xiaoyang; Robe, Eugénie; Jomat, Lucile; Aldon, Didier; Mazars, Christian; Galaud, Jean-Philippe

2017-02-01

Calcium is a universal second messenger involved in various cellular processes including plant development and stress responses. Its conversion into biological responses requires the presence of calcium sensor relays such as calmodulin (CaM) and calmodulin-like (CML) proteins. While the role of CaM is well described, the functions CML proteins remain largely uncharacterized. Here, we show that Arabidopsis CML8 expression is strongly and transiently induced by Pseudomonas syringae, and reverse genetic approaches indicated that the overexpression of CML8 confers on plants a better resistance to pathogenic bacteria compared with wild-type, knock-down and knock-out lines, indicating that CML8 participates as a positive regulator in plant immunity. However, this difference disappeared when inoculations were performed using bacteria unable to inject effectors into a plant host cell or deficient for some effectors known to target the salicylic acid (SA) signaling pathway. SA content and PR1 protein accumulation were altered in CML8 transgenic lines, supporting a role for CML8 in SA-dependent processes. Pathogen-associated molecular pattern (PAMP) treatments with flagellin and elf18 peptides have no effects on CML8 gene expression and do not modify root growth of CML8 knock-down and overexpressing lines compared with wild-type plants. Collectively, our results support a role for CML8 in plant immunity against P. syringae. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

PubMed Central

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 the phytochrome-controlled light response. These results support the hypothesis that calcium functions as a chemical messenger to couple the stimulus of phytochrome photoactivation with physiological responses in plants. Images PMID:11536594

Calcium/Calmodulin-Mediated Gravitropic Response in Plants

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.

2002-01-01

Plant organs respond to different physical signals such as gravity, light and touch. Gravity gives plants proper orientation, resulting in the proper form that we take for granted; the roots grow down into soil and shoots grow towards the light. Under microgravity conditions, as in space, plant growth patterns lack a clear sense of direction. Calcium and calmodulin (CaM) play an important role in gravity signal transduction. However, the molecular and biochemical mechanisms involved in gravity signal transduction are not clearly understood. The goal of this project was to gain a fundamental understanding of how calcium/calmodulin-mediated signaling is involved in gravity signal transduction in plants. During the grant period, significant progress was made in elucidating the role of calmodulin and its target proteins in gravitropism.

Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups.

PubMed

Kelly, Kristen L; Dalton, Shannon R; Wai, Rebecca B; Ramchandani, Kanika; Xu, Rosalind J; Linse, Sara; Londergan, Casey H

2018-03-22

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes' local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin's variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNAmore » is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.« less

Zn2+, not Ca2+, is the most effective cation for activation of dolichol kinase of mammalian brain.

PubMed

Sakakihara, Y; Volpe, J J

1985-12-15

The cation specificity of dolichol kinase of mammalian brain and the potential involvement of a Ca2+-calmodulin system in regulation of this enzyme have been studied. Among 10 divalent cations examined, Zn2+ was found to be most effective for the activation of dolichol kinase of rat and calf brain and cultured C-6 glial cells. The activations with Ca2+, Co2+, and Mg2+ were 53%, 32%, and 18% of the full activation with Zn2+, respectively. No combinations of the cations could activate the enzyme as much as Zn2+ alone. A role for a Ca2+-calmodulin system in the regulation of brain dolichol kinase was not supported by our data. First, the concentration of free Ca2+ required for the maximum activation of dolichol kinase was two to three orders of magnitude greater than the concentration required by typical calmodulin-dependent enzymes. Second, neither the depletion of calmodulin from the microsomal fraction nor the addition of exogenous calmodulin caused an alteration in the activation of dolichol kinase by Ca2+ (or Zn2+). Third, antagonists of calmodulin failed to suppress the activation of the enzyme by Ca2+ (or Zn2+). The data raise the possibility that Zn2+ is involved in the regulation of dolichol kinase in brain.

Regulated expression of a calmodulin isoform alters growth and development in potato.

PubMed

Poovaiah, B W; Takezawa, D; An, G; Han, T J

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails.

PubMed

Holakovska, Blanka; Grycova, Lenka; Bily, Jan; Teisinger, Jan

2011-02-01

The transient receptor potential channels TRPV2 and TRPV5 belong to the vanilloid TRP subfamily. TRPV2 is highly similar to TRPV1 and shares many common properties with it. TRPV5 (and also its homolog TRPV6) is a rather distinct member of the TRPV subfamily. It is distant for being strictly Ca(2+)-selective and features quite different properties from the rest of the TRPV subfamily. It is known that TRP channels are regulated by calmodulin in a calcium-dependent manner. In our study we identified a calmodulin binding site on the C-termini of TRPV2 (654-683) and TRPV5 (587-616) corresponding to the consensus CaM binding motif 1-5-10. The R679 and K681 single mutants of TRPV2 caused a 50% decrease in binding affinity and a double mutation of K661/K664 of the same peptide lowered the binding affinity by up to 75%. A double mutation of R606/K607 and triple mutation of R594/R606/R610 in TRPV5 C-terminal peptide resulted in the total loss of binding affinity to calmodulin. These results demonstrate that the TRPV2 C-tail and TRPV5 C-tail contain calmodulin binding sites and that the basic residues are strongly involved in TRP channel binding to calmodulin.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka

The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical {beta}-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical {beta}-catenin mediated Wnt signalingmore » but also JNK/AP-1 and NF-{kappa}B signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-{kappa}B pathway. Consistent with the central role of Akt, JNK and NF-{kappa}B in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.« less

Quercetin targets the interaction of calcineurin with LxVP-type motifs in immunosuppression

PubMed Central

Zhao, Yane; Zhang, Jin; Shi, Xiaoyu; Li, Jing; Wang, Rui; Song, Ruiwen; Wei, Qun; Cai, Huaibin; Luo, Jing

2016-01-01

Calcineurin (CN) is a unique calcium/calmodulin (CaM)-activated serine/threonine phosphatase. To perform its diverse biological functions, CN communicates with many substrates and other proteins. In the physiological activation of T cells, CN acts through transcriptional factors belonging to the NFAT family and other transcriptional effectors. The classic immunosuppressive drug cyclosporin A (CsA) can bind to cyclophilin (CyP) and compete with CN for the NFAT LxVP motif. CsA has debilitating side effects, including nephrotoxicity, hypertension and tremor. It is desirable to develop alternative immunosuppressive agents. To this end, we first tested the interactions between CN and the LxVP-type substrates, including endogenous regulators of calcineurin (RCAN1) and NFAT. Interestingly, we found that quercetin, the primary dietary flavonol, can inhibit the activity of CN and significantly disrupt the associations between CN and its LxVP-type substrates. We then validated the inhibitory effects of quercetin on the CN-NFAT interactions in cell-based assays. Further, quercetin also shows dose-dependent suppression of cytokine gene expression in mouse spleen cells. These data raise the possibility that the interactions of CN with its LxVP-type substrates are potential targets for immunosuppressive agents. PMID:27109380

An Outbreak of Lymphocutaneous Sporotrichosis among Mine-Workers in South Africa.

PubMed

Govender, Nelesh P; Maphanga, Tsidiso G; Zulu, Thokozile G; Patel, Jaymati; Walaza, Sibongile; Jacobs, Charlene; Ebonwu, Joy I; Ntuli, Sindile; Naicker, Serisha D; Thomas, Juno

2015-09-01

The largest outbreak of sporotrichosis occurred between 1938 and 1947 in the gold mines of Witwatersrand in South Africa. Here, we describe an outbreak of lymphocutaneous sporotrichosis that was investigated in a South African gold mine in 2011. Employees working at a reopened section of the mine were recruited for a descriptive cross-sectional study. Informed consent was sought for interview, clinical examination and medical record review. Specimens were collected from participants with active or partially-healed lymphocutaneous lesions. Environmental samples were collected from underground mine levels. Sporothrix isolates were identified by sequencing of the internal transcribed spacer region of the ribosomal gene and the nuclear calmodulin gene. Of 87 male miners, 81 (93%) were interviewed and examined, of whom 29 (36%) had skin lesions; specimens were collected from 17 (59%). Sporotrichosis was laboratory-confirmed among 10 patients and seven had clinically-compatible lesions. Of 42 miners with known HIV status, 11 (26%) were HIV-infected. No cases of disseminated disease were detected. Participants with ≤ 3 years' mining experience had a four times greater odds of developing sporotrichosis than those who had been employed for >3 years (adjusted OR 4.0, 95% CI 1.2-13.1). Isolates from 8 patients were identified as Sporothrix schenckii sensu stricto by calmodulin gene sequencing while environmental isolates were identified as Sporothrix mexicana. S. schenckii sensu stricto was identified as the causative pathogen. Although genetically distinct species were isolated from clinical and environmental sources, it is likely that the source was contaminated soil and untreated wood underground. No cases occurred following recommendations to close sections of the mine, treat timber and encourage consistent use of personal protective equipment. Sporotrichosis is a potentially re-emerging disease where traditional, rather than heavily mechanised, mining techniques are used. Surveillance should be instituted at sentinel locations.

Calmodulin-like protein 3 is an estrogen receptor alpha coregulator for gene expression and drug response in a SNP, estrogen, and SERM-dependent fashion.

PubMed

Qin, Sisi; Ingle, James N; Liu, Mohan; Yu, Jia; Wickerham, D Lawrence; Kubo, Michiaki; Weinshilboum, Richard M; Wang, Liewei

2017-08-18

We previously performed a case-control genome-wide association study in women treated with selective estrogen receptor modulators (SERMs) for breast cancer prevention and identified single nucleotide polymorphisms (SNPs) in ZNF423 as potential biomarkers for response to SERM therapy. The ZNF423rs9940645 SNP, which is approximately 200 bp away from the estrogen response elements, resulted in the SNP, estrogen, and SERM-dependent regulation of ZNF423 expression and, "downstream", that of BRCA1. Electrophoretic mobility shift assay-mass spectrometry was performed to identify proteins binding to the ZNF423 SNP and coordinating with estrogen receptor alpha (ERα). Clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing was applied to generate ZR75-1 breast cancer cells with different ZNF423 SNP genotypes. Both cultured cells and mouse xenograft models with different ZNF423 SNP genotypes were used to study the cellular responses to SERMs and poly(ADP-ribose) polymerase (PARP) inhibitors. We identified calmodulin-like protein 3 (CALML3) as a key sensor of this SNP and a coregulator of ERα, which contributes to differential gene transcription regulation in an estrogen and SERM-dependent fashion. Furthermore, using CRISPR/Cas9-engineered ZR75-1 breast cancer cells with different ZNF423 SNP genotypes, striking differences in cellular responses to SERMs and PARP inhibitors, alone or in combination, were observed not only in cells but also in a mouse xenograft model. Our results have demonstrated the mechanism by which the ZNF423 rs9940645 SNP might regulate gene expression and drug response as well as its potential role in achieving more highly individualized breast cancer therapy.

Transcriptome Profiling of the Potato (Solanum tuberosum L.) Plant under Drought Stress and Water-Stimulus Conditions.

PubMed

Gong, Lei; Zhang, Hongxia; Gan, Xiaoyan; Zhang, Li; Chen, Yuchao; Nie, Fengjie; Shi, Lei; Li, Miao; Guo, Zhiqian; Zhang, Guohui; Song, Yuxia

2015-01-01

Drought stress can seriously affect tuberization, yield and quality of potato plant. However, the precise molecular mechanisms governing potato stolon's response to drought stress and water supply are not very well understood. In this work, a potato (Solanum tuberosum L.) variant, Ningshu 4, was subjected to severe drought stress treatment (DT) and re-watering treatment (RWT) at tuber bulking stage. Strand-specific cDNA libraries of stolon materials were constructed for paired-end transcriptome sequencing analyses and differentially expressed gene (DEG) examination. In comparison to untreated-control (CT) plants, 3189 and 1797 DEGs were identified in DT and RWT plants and 4154 solely expressed DEGs were screened out from these two comparison groups. Interestingly, 263 genes showed opposite expression patterns in DT and RWT plants. Among them, genes homologous to Protein Phosphatase 2C (PP2C), Aspartic protease in guard cell 1 (ASPG1), auxin-responsive protein, Arabidopsis pseudo response regualtor 2 (APRR2), GA stimulated transcripts in Arabidopsis 6 (GASA6), Calmodulin-like protein 19 (CML19), abscisic acid 8'-hydroxylases and calcium-transporting ATPase, et al. were related with drought-stress and water stimulus response. Sixteen DEGs involved in starch synthesis, accumulation and tuber formation exhibited significantly different expression upon re-watering. In addition, 1630, 1527 and 1596 transcription factor encoding genes were detected in CT, DT and RWT. DEGs of ERF, bHLH, MYB, NAC, WRKY, C2H2, bZIP and HD-ZIP families accounted for 50% in three comparison groups, respectively. Furthermore, characteristics of 565 gene ontology (GO) and 108 Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were analyzed with the 4154 DEGs. All these results suggest that the drought- and water-stimulus response could be implemented by the regulated expression of metabolic pathway DEGs, and these genes were involved in the endogenous hormone biosynthesis and signal transduction pathways. Our data provide more direct information for future study on the interaction between key genes involved in various metabolic pathways under drought stress in potato.

Transcriptome Profiling of the Potato (Solanum tuberosum L.) Plant under Drought Stress and Water-Stimulus Conditions

PubMed Central

Gong, Lei; Zhang, Hongxia; Gan, Xiaoyan; Zhang, Li; Chen, Yuchao; Nie, Fengjie; Shi, Lei; Li, Miao; Guo, Zhiqian; Zhang, Guohui; Song, Yuxia

2015-01-01

Drought stress can seriously affect tuberization, yield and quality of potato plant. However, the precise molecular mechanisms governing potato stolon’s response to drought stress and water supply are not very well understood. In this work, a potato (Solanum tuberosum L.) variant, Ningshu 4, was subjected to severe drought stress treatment (DT) and re-watering treatment (RWT) at tuber bulking stage. Strand-specific cDNA libraries of stolon materials were constructed for paired-end transcriptome sequencing analyses and differentially expressed gene (DEG) examination. In comparison to untreated-control (CT) plants, 3189 and 1797 DEGs were identified in DT and RWT plants and 4154 solely expressed DEGs were screened out from these two comparison groups. Interestingly, 263 genes showed opposite expression patterns in DT and RWT plants. Among them, genes homologous to Protein Phosphatase 2C (PP2C), Aspartic protease in guard cell 1 (ASPG1), auxin-responsive protein, Arabidopsis pseudo response regualtor 2 (APRR2), GA stimulated transcripts in Arabidopsis 6 (GASA6), Calmodulin-like protein 19 (CML19), abscisic acid 8'-hydroxylases and calcium-transporting ATPase, et al. were related with drought-stress and water stimulus response. Sixteen DEGs involved in starch synthesis, accumulation and tuber formation exhibited significantly different expression upon re-watering. In addition, 1630, 1527 and 1596 transcription factor encoding genes were detected in CT, DT and RWT. DEGs of ERF, bHLH, MYB, NAC, WRKY, C2H2, bZIP and HD-ZIP families accounted for 50% in three comparison groups, respectively. Furthermore, characteristics of 565 gene ontology (GO) and 108 Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were analyzed with the 4154 DEGs. All these results suggest that the drought- and water-stimulus response could be implemented by the regulated expression of metabolic pathway DEGs, and these genes were involved in the endogenous hormone biosynthesis and signal transduction pathways. Our data provide more direct information for future study on the interaction between key genes involved in various metabolic pathways under drought stress in potato. PMID:26010543

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

PubMed Central

Neumüller, Klaus G.; Elsayad, Kareem; Reisecker, Johannes M.; Waxham, M. Neal; Heinze, Katrin G.

2010-01-01

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. PMID:21124984

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

PubMed Central

D'Angelo, Maximiliano A; Sanguineti, Santiago; Reece, Jeffrey M; Birnbaumer, Lutz; Torres, Héctor N; Flawiá, Mirtha M

2004-01-01

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites. PMID:14556647

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

NASA Technical Reports Server (NTRS)

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.

Hydrophobic Peptides Affect Binding of Calmodulin and Ca2+ as Explored by H/D Amide Exchange and Mass Spectrometry

PubMed Central

Sperry, Justin B.; Huang, Richard Y-C.; Zhu, Mei M.; Rempel, Don L.; Gross, Michael L.

2010-01-01

Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high resolution PLIMSTEX (Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca2+, the extent of HDX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-saturated calmodulin exhibits increased protection when bound to the peptides, forming high affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca2+. Titration of the protein using PLIMSTEX provides the binding affinity of Ca2+ to calmodulin within previously reported values. The affinities of calmodulin to Ca2+ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The titration data taken in this way show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca2+-binding properties of this well-studied protein. PMID:21765646

Structure and dynamics of calmodulin in solution.

PubMed Central

Wriggers, W; Mehler, E; Pitici, F; Weinstein, H; Schulten, K

1998-01-01

To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides. PMID:9545028

Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Radogna, F.; Sestili, P.; Martinelli, C.

We have shown that melatonin immediately and transiently stimulates intracellular free radical production on a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and are accompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation of AA; inhibition of Ca{sup 2+}-independent, but not Ca{sup 2+}-dependent, phospholipase A2 (PLA2), prevents both melatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS, indicating that PLA2 is upstream with respect to LOX, asmore » occurs in many signaling pathways. Chlorpromazine, an inhibitor of melatonin-calmodulin interaction, inhibits both ROS and arachidonic acid production, thus possibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it is known that Ca{sup 2+}-independent PLA2 binds to calmodulin: our results are compatible with PLA2 being liberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signal transduction. These results delineate a novel molecular pathway through which melatonin may participate to the inflammatory response.« less

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology

PubMed Central

Wieschhaus, Adam; Khan, Anwar; Zaidi, Asma; Rogalin, Henry; Hanada, Toshihiko; Liu, Fei; De Franceschi, Lucia; Brugnara, Carlo; Rivera, Alicia; Chishti, Athar H.

2014-01-01

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+–Cl− co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease. PMID:22870887

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

PubMed

Wieschhaus, Adam; Khan, Anwar; Zaidi, Asma; Rogalin, Henry; Hanada, Toshihiko; Liu, Fei; De Franceschi, Lucia; Brugnara, Carlo; Rivera, Alicia; Chishti, Athar H

2012-11-15

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

PubMed

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

Evaluation of plasma membrane calcium/calmodulin-dependent ATPase isoform 4 as a potential target for fertility control.

PubMed

Cartwright, Elizabeth J; Neyses, Ludwig

2010-01-01

The array of contraceptives currently available is clearly inadequate and does not meet consumer demands since it is estimated that up to a quarter of all pregnancies worldwide are unintended. There is, therefore, an overwhelming global need to develop new effective, safe, ideally non-hormonal contraceptives for both male and female use. The contraceptive field, unlike other areas such as cancer, has a dearth of new targets. We have addressed this issue and propose that isoform 4 of the plasma membrane calcium ATPase is a potentially exciting novel target for fertility control. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function in the majority of cells is to extrude calcium to the extracellular milieu. Two isoforms of this gene family, PMCA1 and PMCA4, are expressed in spermatozoa, with PMCA4 being the predominant isoform. Although this gene is ubiquitously expressed, its function is highly tissue-specific. Genetic deletion of PMCA4, in PMCA4 knockout mice, led to 100% infertility specifically in the male mutant mice due to a selective defect in sperm motility. It is important to note that the gene deletion did not affect normal mating characteristics in these mice. This phenotype was mimicked in wild-type sperm treated with the non-specific PMCA inhibitor 5-(and 6-) carboxyeosin diacetate succinimidyl ester; a proof-of-principle that inhibition of PMCA4 has potential importance in the control of fertility. This review outlines the potential for PMCA4 to be a novel target for fertility control by acting to inhibit sperm motility. It will outline the characteristics that make this target drugable and will describe methodologies to identify and validate novel inhibitors of this target.

Calmodulin-regulated adenylyl cyclases and neuromodulation.

PubMed

Xia, Z; Storm, D R

1997-06-01

Coincidence detection and crosstalk between signal transduction systems play very important regulatory roles in the nervous system, particularly in the regulation of transcription. Coupling of the Ca2+ and cAMP regulatory systems by calmodulin-regulated adenylyl cyclases is hypothesized to be important for some forms of synaptic plasticity, neuroendocrine function, and olfactory detection. Recent studies of a mutant mouse deficient in type I calmodulin-sensitive adenylyl cyclase have provided the first evidence that adenylyl cyclases are important for synaptic plasticity, as well as for learning and memory in vertebrates.

Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2A receptor.

PubMed

Piirainen, Henni; Taura, Jaume; Kursula, Petri; Ciruela, Francisco; Jaakola, Veli-Pekka

2017-04-01

Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A 2A receptor (A 2A R), has an exceptionally long intracellular C terminus (A 2A R-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to A 2A R and the role of Ca 2+ in this process. First, we studied the A 2A R-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the A 2A R-ct through its distal calmodulin-like domain in a Ca 2+ -independent manner with a dissociation constant of 5-12μM, thus showing an ~100 times lower affinity compared to the A 2A R-calmodulin/Ca 2+ complex. Importantly, calmodulin displaced α-actinin 1 from the A 2A R-ct in a Ca 2+ -dependent fashion, disrupting the A 2A R-α-actinin 1 complex. Finally, we assessed the impact of Ca 2+ on A 2A R internalization in living cells, a function operated by the A 2A R-α-actinin 1 complex. Interestingly, while Ca 2+ influx did not affect constitutive A 2A R endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A 2A R/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A 2A R with calmodulin and α-actinin 1 is fine-tuned by Ca 2+ , a fact that might power agonist-mediated receptor internalization and function. Copyright © 2017 Elsevier B.V. All rights reserved.

Calmodulin permanently associates with rat olfactory CNG channels under native conditions.

PubMed

Bradley, Jonathan; Bönigk, Wolfgang; Yau, King-Wai; Frings, Stephan

2004-07-01

An important mechanism by which vertebrate olfactory sensory neurons rapidly adapt to odorants is feedback modulation of the Ca(2+)-permeable cyclic nucleotide-gated (CNG) transduction channels. Extensive heterologous studies of homomeric CNGA2 channels have led to a molecular model of channel modulation based on the binding of calcium-calmodulin to a site on the cytoplasmic amino terminus of CNGA2. Native rat olfactory CNG channels, however, are heteromeric complexes of three homologous but distinct subunits. Notably, in heteromeric channels, we found no role for CNGA2 in feedback modulation. Instead, an IQ-type calmodulin-binding site on CNGB1b and a similar but previously unidentified site on CNGA4 are necessary and sufficient. These sites seem to confer binding of Ca(2+)-free calmodulin (apocalmodulin), which is then poised to trigger inhibition of native channels in the presence of Ca(2+).

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels.

PubMed

Tobelaim, William S; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E; Hirsch, Joel A; Attali, Bernard

2017-11-02

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow I KS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and recently we revealed the competition of PIP 2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP 2 and Ca 2+ -CaM perform the same function on I KS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca 2+ -CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.

Species of Cercospora associated with grey leaf spot of maize

PubMed Central

Crous, Pedro W.; Groenewald, Johannes Z.; Groenewald, Marizeth; Caldwell, Pat; Braun, Uwe; Harrington, Thomas C.

2006-01-01

Grey leaf spot is a serious yield-reducing disease of maize (Zea mays) in many parts of the world where this crop is cultivated. The causal organism associated with the disease is Cercospora zeae-maydis. Two potential sibling species have been recognized as Groups I and II. The DNA sequences for the internal transcribed spacers (ITS1 & ITS2), the 5.8S rRNA gene, elongation factor 1-α, histone H3, actin and calmodulin gene regions suggest that Groups I and II are two distinct species. Furthermore, Cercospora zeae-maydis (Group I) can be distinguished from C. zeina sp. nov. (Group II) by its faster growth rate on artificial media, the ability to produce cercosporin, longer conidiophores, and broadly fusiform conidia. A PCR-based test that distinguishes the two species was developed using species-specific primers designed from the histone H3 gene. PMID:18490979

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Changes in calmodulin concentration and cyclic 3',5'-nucleotide phosphodiesterase activity in skeletal muscle of hyper- and hypothyroid rats.

PubMed

Mano, T; Iwase, K; Yoshimochi, I; Sawai, Y; Oda, N; Nishida, Y; Mokuno, T; Kotake, M; Nakai, A; Hayakawa, N

1995-08-01

Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.

Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin.

PubMed

Ambrosino, Paolo; Alaimo, Alessandro; Bartollino, Silvia; Manocchio, Laura; De Maria, Michela; Mosca, Ilaria; Gomis-Perez, Carolina; Alberdi, Araitz; Scambia, Giovanni; Lesca, Gaetan; Villarroel, Alvaro; Taglialatela, Maurizio; Soldovieri, Maria Virginia

2015-09-01

Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K(+) channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca(2+)). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies. Copyright © 2015 Elsevier B.V. All rights reserved.

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.

PubMed Central

Chen, M X; Bouquin, N; Norris, V; Casarégola, S; Séror, S J; Holland, I B

1991-01-01

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein. Images PMID:1915285

Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

PubMed

Piirainen, Henni; Hellman, Maarit; Tossavainen, Helena; Permi, Perttu; Kursula, Petri; Jaakola, Veli-Pekka

2015-02-17

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

ATRX, a member of the SNF2 family of helicase/ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes.

PubMed

De La Fuente, Rabindranath; Viveiros, Maria M; Wigglesworth, Karen; Eppig, John J

2004-08-01

ATRX is a centromeric heterochromatin binding protein belonging to the SNF2 family of helicase/ATPases with chromatin remodeling activity. Mutations in the human ATRX gene result in X-linked alpha-thalassaemia with mental retardation (ATRX) syndrome and correlate with changes in methylation of repetitive DNA sequences. We show here that ATRX also functions to regulate key stages of meiosis in mouse oocytes. At the germinal vesicle (GV) stage, ATRX was found associated with the perinucleolar heterochromatin rim in transcriptionally quiescent oocytes. Phosphorylation of ATRX during meiotic maturation is dependent upon calcium calmodulin kinase (CamKII) activity. Meiotic resumption also coincides with deacetylation of histone H4 at lysine 5 (H4K5 Ac) while ATRX and histone H3 methylated on lysine 9 (H3K9) remained bound to the centromeres and interstitial regions of condensing chromosomes, respectively. Inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) disrupted ATRX binding to the centromeres of hyperacetylated chromosomes resulting in abnormal chromosome alignments at metaphase II (MII). Similarly, while selective ablation of ATRX by antibody microinjection and RNA interference (RNAi) had no effect on the progression of meiosis, it had severe consequences for the alignment of chromosomes on the metaphase II spindle. These results suggest that genome-wide epigenetic modifications such as global histone deacetylation are essential for the binding of ATRX to centromeric heterochromatin. Moreover, centromeric ATRX is required for correct chromosome alignment and organization of a bipolar meiotic metaphase II spindle.

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

PubMed

Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

2017-01-01

Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

Olfactory CNG channel desensitization by Ca2+/CaM via the B1b subunit affects response termination but not sensitivity to recurring stimulation.

PubMed

Song, Yijun; Cygnar, Katherine D; Sagdullaev, Botir; Valley, Matthew; Hirsh, Sarah; Stephan, Aaron; Reisert, Johannes; Zhao, Haiqing

2008-05-08

Ca2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses.

Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca[superscript 2+]·calmodulins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fallon, Jennifer L.; Baker, Mariah R.; Xiong, Liangwen

2009-11-10

Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is anmore » unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.« less

Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity.

PubMed

Evans, D J; Frank, D W; Finck-Barbançon, V; Wu, C; Fleiszig, S M

1998-04-01

Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

Characterization of diabetic nephropathy in CaM kinase IIalpha (Thr286Asp) transgenic mice.

PubMed

Suzuki, Hikari; Kato, Ichiro; Usui, Isao; Takasaki, Ichiro; Tabuchi, Yoshiaki; Oya, Takeshi; Tsuneyama, Koichi; Kawaguchi, Hiroshi; Hiraga, Koichi; Takasawa, Shin; Okamoto, Hiroshi; Tobe, Kazuyuki; Sasahara, Masakiyo

2009-01-30

Detailed studies were performed on diabetic kidneys derived from transgenic mice overexpressing the mutant form (Thr286Asp) of Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM kinase IIalpha) in pancreatic beta-cells. Kidney weight/body weight ratio, urinary albumin/creatinine ratio, serum BUN level, and mesangial/glomerular area ratio were all significantly higher in transgenic mice than in wild-type mice. cDNA microarray analysis revealed 17 up-regulated genes and 12 down-regulated genes in transgenic kidney. Among up-regulated genes, cyclin D2 (6.70-fold) and osteopontin (2.35-fold) were thought to play important roles in the progression of diabetic nephropathy. Transgenic glomeruli and tubular epithelial cells were strongly stained for osteopontin, a molecule which induces immune response. In quantitative real-time RT-PCR analyses, expressions of not only M1 macrophage marker genes but also M2 macrophage marker genes were elevated in renal cortex of transgenic mice. Overall results indicate that CaM kinase IIalpha (Thr286Asp) transgenic mice serve as an excellent model for diabetic nephropathy.

Inhibition of human calcineurin and yeast calcineurin-dependent gene expression by Jasminum humile leaf and root extracts.

PubMed

Prescott, Thomas A K; Ariño, Joaquín; Kite, Geoffrey C; Simmonds, Monique S J

2012-03-27

The leaves of Jasminum humile are used to treat skin disorders in a way which resembles the use of modern topical anti-inflammatory drugs. Ethanolic extracts of the roots and leaves were shown to inhibit calcineurin which is a regulator of inflammatory gene expression. A novel yeast calcineurin reporter gene assay suitable for a 96 well plate format was developed to test for inhibition of calcineurin-dependent gene expression. Calmodulin/calcineurin phosphatase assays were then used to further elucidate the mode of action of the extracts. Jasminum humile root and leaf extract exhibited calcineurin inhibition activity that was shown to be mediated through a direct interaction with calcineurin enzyme. The activity is sufficient to block calcineurin-dependent gene expression in a yeast model. The activity of the plant supports its traditional use in the treatment of inflammatory skin disorders. The specially adapted yeast reporter assay was found to be a highly effective way of detecting calcineurin inhibitors in plant extracts. Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

Phospholipase D inhibition by hexanal is associated with calcium signal transduction events in raspberry

PubMed Central

Kayal, Walid El; Paliyath, Gopinadhan; Sullivan, J Alan; Subramanian, Jayasankar

2017-01-01

Raspberry (Rubus spp.) is an economically important crop with a restricted growing season and very limited fruit shelf-life due to its extreme tenderness. In order to prolong its shelf life, an aqueous composition containing hexanal as the key active ingredient (HC) was applied as a preharvest spray during fruit development. The effects of HC were assessed using physiological, biochemical and anatomical parameters on the treated fruits and compared with the effects of mock inoculation which lacked hexanal. Sugars and acidity did not show a significant change in response to HC treatment, while the pulling force (the tension required to detach the berry from the receptacle) significantly improved in the HC-treated fruits, compared to control. Scanning electron microscope (SEM) analysis revealed a high correlation between the presence of rigid epidermal hairs and a stronger degree of attachment between berries and their receptacle in the HC treated fruits. Further, electron micrographs also showed abnormal crystalline depositions on the epidermal drupelets of the treated berries. Energy Dispersive X-ray Spectroscopy (EDS) analysis showed those crystals to be largely composed of calcium. HC treatment also resulted in the reduction of transcript level of three phospholipase D genes, as well as altered expression pattern of five members of the annexin gene family, and four calmodulin-binding transcription activators. Quantification of PLD activity showed that hexanal inhibited PLD activity in treated berries. The potential crosstalk between hexanal, phospholipase D activity and calcium and this crosstalk’s role in delaying fruit softening and in prolonging storage life of fruits shelf life is discussed. PMID:29114390

Social feeding in Caenorhabditis elegans is modulated by antipsychotic drugs and calmodulin and may serve as a protophenotype for asociality.

PubMed

Dwyer, Donard S; Awatramani, Poonam; Thakur, Rashmi; Seeni, Ramya; Aamodt, Eric J

2015-05-01

Here, we define a protophenotype as an endophenotype that has been conserved during evolution. Social feeding in Caenorhabditis elegans may be an example of a protophenotype related to asociality in schizophrenia. It is regulated by the highly conserved neuropeptide Y receptor, NPR-1, and we speculated that social feeding should be affected by antipsychotic drugs. The social feeding strain, npr-1(g320), was exposed to antipsychotic drugs, dopamine or calmodulin antagonists on plates with bacterial lawns, and the number of aggregates on the plates was counted as a measure of social feeding. First-generation antipsychotics, chlorpromazine, trifluoperazine, fluphenazine, and haloperidol, and the second-generation drug, olanzapine, inhibited social feeding. Dopamine accelerated aggregation, whereas selective D2 dopamine receptor antagonists, sulpiride and raclopride, were inhibitory. Calmodulin antagonists effectively inhibited social feeding, as did RNAi knockdown of calmodulin (cmd-1) expression. In addition, gap junction inhibitors prevented aggregation, which is consistent with the hub-and-spoke arrangement of neurons that regulate social feeding via functional gap junctions. The studies described here revealed novel connections between dopaminergic signaling, the NPY receptor, calmodulin, and gap junctions in the regulation of social behavior in C. elegans. These pathways are evolutionarily-conserved, and have also been implicated in the pathogenesis of schizophrenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transient dissociation of polyribosomes and concurrent recruitment of calreticulin and calmodulin transcripts in gravistimulated maize pulvini

NASA Technical Reports Server (NTRS)

Heilmann, I.; Shin, J.; Huang, J.; Perera, I. Y.; Davies, E.

2001-01-01

The dynamics of polyribosome abundance were studied in gravistimulated maize (Zea mays) stem pulvini. During the initial 15 min of gravistimulation, the amount of large polyribosomes transiently decreased. The transient decrease in polyribosome levels was accompanied by a transient decrease in polyribosome-associated mRNA. After 30 min of gravistimulation, the levels of polyribosomes and the amount of polyribosome-associated mRNA gradually increased over 24 h up to 3- to 4-fold of the initial value. Within 15 min of gravistimulation, total levels of transcripts coding for calreticulin and calmodulin were elevated 5-fold in maize pulvinus total RNA. Transcripts coding for calreticulin and calmodulin were recruited into polyribosomes within 15 min of gravistimulation. Over 4 h of gravistimulation, a gradual increase in the association of calreticulin and calmodulin transcripts with polyribosomes was seen predominantly in the lower one-half of the maize pulvinus; the association of transcripts for vacuolar invertase with polyribosomes did not change over this period. Our results suggest that within 15 min of gravistimulation, the translation of the majority of transcripts associated with polyribosomes decreased, resembling a general stress response. Recruitment of calreticulin and calmodulin transcripts into polyribosomes occurred predominantly in the lower pulvinus one-half during the first 4 h when the presentation time for gravistimulation in the maize pulvinus is not yet complete.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

NASA Technical Reports Server (NTRS)

Safadi, F.; Reddy, V. S.; Reddy, A. S.

2000-01-01

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

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

EPA Science Inventory

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...

Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya

PubMed Central

Samson, R.A.; Hong, S.; Peterson, S.W.; Frisvad, J.C.; Varga, J.

2007-01-01

The taxonomy of Aspergillus section Fumigati with its teleomorph genus Neosartorya is revised. The species concept is based on phenotypic (morphology and extrolite profiles) and molecular (β-tubulin and calmodulin gene sequences) characters in a polyphasic approach. Four new taxa are proposed: N. australensis N. ferenczii, N. papuaensis and N. warcupii. All newly described and accepted species are illustrated. The section consists of 33 taxa: 10 strictly anamorphic Aspergillus species and 23 Neosartorya species. Four other Neosartorya species described previously were not available for this monograph, and consequently are relegated to the category of doubtful species. PMID:18490953

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

PubMed Central

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

2015-01-01

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 CaM KMT gene. PMID:26247364

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

PubMed

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 CaM KMT gene.

An Outbreak of Lymphocutaneous Sporotrichosis among Mine-Workers in South Africa

PubMed Central

Govender, Nelesh P.; Maphanga, Tsidiso G.; Zulu, Thokozile G.; Patel, Jaymati; Walaza, Sibongile; Jacobs, Charlene; Ebonwu, Joy I.; Ntuli, Sindile; Naicker, Serisha D.; Thomas, Juno

2015-01-01

Background The largest outbreak of sporotrichosis occurred between 1938 and 1947 in the gold mines of Witwatersrand in South Africa. Here, we describe an outbreak of lymphocutaneous sporotrichosis that was investigated in a South African gold mine in 2011. Methodology Employees working at a reopened section of the mine were recruited for a descriptive cross-sectional study. Informed consent was sought for interview, clinical examination and medical record review. Specimens were collected from participants with active or partially-healed lymphocutaneous lesions. Environmental samples were collected from underground mine levels. Sporothrix isolates were identified by sequencing of the internal transcribed spacer region of the ribosomal gene and the nuclear calmodulin gene. Principal Findings Of 87 male miners, 81 (93%) were interviewed and examined, of whom 29 (36%) had skin lesions; specimens were collected from 17 (59%). Sporotrichosis was laboratory-confirmed among 10 patients and seven had clinically-compatible lesions. Of 42 miners with known HIV status, 11 (26%) were HIV-infected. No cases of disseminated disease were detected. Participants with ≤3 years’ mining experience had a four times greater odds of developing sporotrichosis than those who had been employed for >3 years (adjusted OR 4.0, 95% CI 1.2–13.1). Isolates from 8 patients were identified as Sporothrix schenckii sensu stricto by calmodulin gene sequencing while environmental isolates were identified as Sporothrix mexicana. Conclusions/Significance S. schenckii sensu stricto was identified as the causative pathogen. Although genetically distinct species were isolated from clinical and environmental sources, it is likely that the source was contaminated soil and untreated wood underground. No cases occurred following recommendations to close sections of the mine, treat timber and encourage consistent use of personal protective equipment. Sporotrichosis is a potentially re-emerging disease where traditional, rather than heavily mechanised, mining techniques are used. Surveillance should be instituted at sentinel locations. PMID:26407300

Development and molecular characterization of genic molecular markers for grain protein and calcium content in finger millet (Eleusine coracana (L.) Gaertn.).

PubMed

Nirgude, M; Babu, B Kalyana; Shambhavi, Y; Singh, U M; Upadhyaya, H D; Kumar, Anil

2014-03-01

Finger millet (Eleusine coracana (L.) Gaertn), holds immense agricultural and economic importance for its high nutraceuticals quality. Finger millets seeds are rich source of calcium and its proteins are good source of essential amino acids. In the present study, we developed 36 EST-SSR primers for the opaque2 modifiers and 20 anchored-SSR primers for calcium transporters and calmodulin for analysis of the genetic diversity of 103 finger millet genotypes for grain protein and calcium contents. Out of the 36 opaque2 modifiers primers, 15 were found polymorphic and were used for the diversity analysis. The highest PIC value was observed with the primer FMO2E33 (0.26), while the lowest was observed FMO2E27 (0.023) with an average value of 0.17. The gene diversity was highest for the primer FMO2E33 (0.33), however it was lowest for FMO2E27 (0.024) at average value of 0.29. The percentage polymorphism shown by opaque2 modifiers primers was 68.23%. The diversity analysis by calcium transporters and calmodulin based anchored SSR loci revealed that the highest PIC was observed with the primer FMCA8 (0.30) and the lowest was observed for FMCA5 (0.023) with an average value of 0.18. The highest gene diversity was observed for primer FMCA8 (0.37), while lowest for FMCA5 (0.024) at an average of 0.21. The opaque2 modifiers specific EST-SSRs could able to differentiate the finger millet genotypes into high, medium and low protein containing genotypes. However, calcium dependent candidate gene based EST-SSRs could broadly differentiate the genotypes based on the calcium content with a few exceptions. A significant negative correlation between calcium and protein content was observed. The present study resulted in identification of highly polymorphic primers (FMO2E30, FMO2E33, FMO2-18 and FMO2-14) based on the parameters such as percentage of polymorphism, PIC values, gene diversity and number of alleles.

Absolute configuration of acremoxanthone C, a potent calmodulin inhibitor from Purpureocillium lilacinum

USDA-ARS?s Scientific Manuscript database

Bioassay-guided fractionation of an extract prepared from the culture medium and mycelium of Purpureocillium lilacinum allowed the isolation of two calmodulin (CaM) inhibitors, namely, acremoxanthone C (1) and acremonidin A (2). The absolute configuration of 1 was established as 2R, 3R, 1'S, 11'S, ...

The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekiya, M.; Frohlich, E.D.; Cole, F.E.

1991-01-01

In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation.more » In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.« less

Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

DOE PAGES

Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.; ...

2016-03-07

Activation triggers the exchange of subunits in Ca 2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This convertsmore » the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.« less

Expression and affinity purification of recombinant proteins from plants

NASA Technical Reports Server (NTRS)

Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

2002-01-01

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

Calcium transport in vesicles from carrot cells: Stimulation by calmodulin and phosphatidylserine. [Daucus carota cv. Danvers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenling Hsieh; Sze, Heven

1991-05-01

The transport properties of Ca-pumping ATPases from carrot (Daucus carota cv. Danvers) tissue culture cells were studied. ATP dependent Ca transport in vesicles that comigrated with an ER marker, was stimulated 3-4 fold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca-ATPase) partially inhibited oxalate-stimulated Ca transport activity; however, it had little or not effect on calmodulin-stimulated Ca uptake. The results suggested the presence of two types of Ca ATPases, and ER- and a plasma membrane-type. Incubation of membranes with (gamma{sup 32}P)ATP resulted in the formation of a single acyl ({sup 32}P) phosphoprotein of 120 kDa. Formationmore » of this phosphoprotein was dependent on Ca, and enhanced by La {sup 3+}, characteristic of the plasma membrane CaATPase. Acidic phospholipids, like phosphatidylserine, stimulated Ca transport, similar to their effect on the erythrocyte plasma membrane CaATPase. These results would indicate that the calmodulin-stimulated Ca transport originated in large part from a plasma membrane-type Ca pump of 120 kDa.« less

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

PubMed

Bitar, K N; Hillemeier, C; Biancani, P

1990-01-01

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.

Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.

Activation triggers the exchange of subunits in Ca 2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This convertsmore » the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.« less

Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II

PubMed Central

Bhattacharyya, Moitrayee; Stratton, Margaret M; Going, Catherine C; McSpadden, Ethan D; Huang, Yongjian; Susa, Anna C; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L; Barros, Tiago; Schulman, Howard; Williams, Evan R; Kuriyan, John

2016-01-01

Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. DOI: http://dx.doi.org/10.7554/eLife.13405.001 PMID:26949248

Short- and long-term inhibition of cardiac inward-rectifier potassium channel current by an antiarrhythmic drug bepridil.

PubMed

Ma, Fangfang; Takanari, Hiroki; Masuda, Kimiko; Morishima, Masaki; Ono, Katsushige

2016-07-01

Bepridil is an effective antiarrhythmic drug on supraventricular and ventricular arrhythmias, and inhibitor of calmodulin. Recent investigations have been elucidating that bepridil exerts antiarrhythmic effects through its acute and chronic application for patients. The aim of this study was to identify the efficacy and the potential mechanism of bepridil on the inward-rectifier potassium channel in neonatal rat cardiomyocytes in acute- and long-term conditions. Bepridil inhibited inward-rectifier potassium current (I K1) as a short-term effect with IC50 of 17 μM. Bepridil also reduced I K1 of neonatal cardiomyocytes when applied for 24 h in the culture medium with IC50 of 2.7 μM. Both a calmodulin inhibitor (W-7) and an inhibitor of calmodulin-kinase II (KN93) reduced I K1 when applied for 24 h as a long-term effect in the same fashion, suggesting that the long-term application of bepridil inhibits I K1 more potently than that of the short-term application through the inhibition of calmodulin kinase II pathway in cardiomyocytes.

Molecular cloning and evolutionary analysis of the calcium-modulated contractile protein, centrin, in green algae and land plants.

PubMed

Bhattacharya, D; Steinkötter, J; Melkonian, M

1993-12-01

Centrin (= caltractin) is a ubiquitous, cytoskeletal protein which is a member of the EF-hand superfamily of calcium-binding proteins. A centrin-coding cDNA was isolated and characterized from the prasinophyte green alga Scherffelia dubia. Centrin PCR amplification primers were used to isolate partial, homologous cDNA sequences from the green algae Tetraselmis striata and Spermatozopsis similis. Annealing analyses suggested that centrin is a single-copy-coding region in T. striata and S. similis and other green algae studied. Centrin-coding regions from S. dubia, S. similis and T. striata encode four colinear EF-hand domains which putatively bind calcium. Phylogenetic analyses, including homologous sequences from Chlamydomonas reinhardtii and the land plant Atriplex nummularia, demonstrate that the domains of centrins are congruent and arose from the two-fold duplication of an ancestral EF hand with Domains 1+3 and Domains 2+4 clustering. The domains of centrins are also congruent with those of calmodulins demonstrating that, like calmodulin, centrin is an ancient protein which arose within the ancestor of all eukaryotes via gene duplication. Phylogenetic relationships inferred from centrin-coding region comparisons mirror results of small subunit ribosomal RNA sequence analyses suggesting that centrin-coding regions are useful evolutionary markers within the green algae.

Native and engineered sensors for Ca2+ and Zn2+: lessons from calmodulin and MTF1.

PubMed

Carpenter, Margaret C; Palmer, Amy E

2017-05-09

Ca 2+ and Zn 2+ dynamics have been identified as important drivers of physiological processes. In order for these dynamics to encode function, the cell must have sensors that transduce changes in metal concentration to specific downstream actions. Here we compare and contrast the native metal sensors: calmodulin (CaM), the quintessential Ca 2+ sensor and metal-responsive transcription factor 1 (MTF1), a candidate Zn 2+ sensor. While CaM recognizes and modulates the activity of hundreds of proteins through allosteric interactions, MTF1 recognizes a single DNA motif that is distributed throughout the genome regulating the transcription of many target genes. We examine how the different inorganic chemistries of these two metal ions may shape these different mechanisms transducing metal ion concentration into changing physiologic activity. In addition to native metal sensors, scientists have engineered sensors to spy on the dynamic changes of metals in cells. The inorganic chemistry of the metals shapes the possibilities in the design strategies of engineered sensors. We examine how different strategies to tune the affinities of engineered sensors mirror the strategies nature developed to sense both Ca 2+ and Zn 2+ in cells. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Calcineurin-Crz1 Signaling in Lower Eukaryotes

PubMed Central

2014-01-01

Calcium ions are ubiquitous intracellular messengers. An increase in the cytosolic Ca2+ concentration activates many proteins, including calmodulin and the Ca2+/calmodulin-dependent protein phosphatase calcineurin. The phosphatase is conserved from yeast to humans (except in plants), and many target proteins of calcineurin have been identified. The most prominent and best-investigated targets, however, are the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeast. In recent years, many orthologues of Crz1 have been identified and characterized in various species of fungi, amoebae, and other lower eukaryotes. It has been shown that the functions of calcineurin-Crz1 signaling, ranging from ion homeostasis through cell wall biogenesis to the building of filamentous structures, are conserved in the different organisms. Furthermore, frequency-modulated gene expression through Crz1 has been discovered as a striking new mechanism by which cells can coordinate their response to a signal. In this review, I focus on the latest findings concerning calcineurin-Crz1 signaling in fungi, amoebae and other lower eukaryotes. I discuss the potential of Crz1 and its orthologues as putative drug targets, and I also discuss possible parallels with calcineurin-NFAT signaling in mammals. PMID:24681686

Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion.

PubMed

Cao, Qi; Zhong, Xi Zoë; Zou, Yuanjie; Murrell-Lagnado, Ruth; Zhu, Michael X; Dong, Xian-Ping

2015-06-22

Intra-endolysosomal Ca(2+) release is required for endolysosomal membrane fusion with intracellular organelles. However, the molecular mechanisms for intra-endolysosomal Ca(2+) release and the downstream Ca(2+) targets involved in the fusion remain elusive. Previously, we demonstrated that endolysosomal P2X4 forms channels activated by luminal adenosine triphosphate in a pH-dependent manner. In this paper, we show that overexpression of P2X4, as well as increasing endolysosomal P2X4 activity by alkalinization of endolysosome lumen, promoted vacuole enlargement in cells and endolysosome fusion in a cell-free assay. These effects were prevented by inhibiting P2X4, expressing a dominant-negative P2X4 mutant, and disrupting the P2X4 gene. We further show that P2X4 and calmodulin (CaM) form a complex at endolysosomal membrane where P2X4 activation recruits CaM to promote fusion and vacuolation in a Ca(2+)-dependent fashion. Moreover, P2X4 activation-triggered fusion and vacuolation were suppressed by inhibiting CaM. Our data thus suggest a new molecular mechanism for endolysosomal membrane fusion involving P2X4-mediated endolysosomal Ca(2+) release and subsequent CaM activation. © 2015 Cao et al.

Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

PubMed Central

Cao, Qi; Zhong, Xi Zoë; Zou, Yuanjie; Murrell-Lagnado, Ruth; Zhu, Michael X.

2015-01-01

Intra-endolysosomal Ca2+ release is required for endolysosomal membrane fusion with intracellular organelles. However, the molecular mechanisms for intra-endolysosomal Ca2+ release and the downstream Ca2+ targets involved in the fusion remain elusive. Previously, we demonstrated that endolysosomal P2X4 forms channels activated by luminal adenosine triphosphate in a pH-dependent manner. In this paper, we show that overexpression of P2X4, as well as increasing endolysosomal P2X4 activity by alkalinization of endolysosome lumen, promoted vacuole enlargement in cells and endolysosome fusion in a cell-free assay. These effects were prevented by inhibiting P2X4, expressing a dominant-negative P2X4 mutant, and disrupting the P2X4 gene. We further show that P2X4 and calmodulin (CaM) form a complex at endolysosomal membrane where P2X4 activation recruits CaM to promote fusion and vacuolation in a Ca2+-dependent fashion. Moreover, P2X4 activation-triggered fusion and vacuolation were suppressed by inhibiting CaM. Our data thus suggest a new molecular mechanism for endolysosomal membrane fusion involving P2X4-mediated endolysosomal Ca2+ release and subsequent CaM activation. PMID:26101220

Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation.

PubMed

Taschner, Michael J; Rafigh, Mehran; Lampert, Fabienne; Schnaiter, Simon; Hartmann, Christine

2008-05-01

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.

Cancer stem cell drugs target K-ras signaling in a stemness context

PubMed Central

Najumudeen, A K; Jaiswal, A; Lectez, B; Oetken-Lindholm, C; Guzmán, C; Siljamäki, E; Posada, I M D; Lacey, E; Aittokallio, T; Abankwa, D

2016-01-01

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC. PMID:26973241

High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.

PubMed

Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos

2017-05-13

Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

β-Adducin siRNA disruption of the spectrin-based cytoskeleton in differentiating keratinocytes prevented by calcium acting through calmodulin/epidermal growth factor receptor/cadherin pathway.

PubMed

Wu, Jianghong; Masci, Paul P; Chen, Chenfeng; Chen, Jiezhong; Lavin, Martin F; Zhao, Kong-Nan

2015-01-01

Here, we report that siRNA transfection of β-adducin significantly disrupted the spectrin-based cytoskeleton and cytoskeletal arrangements of both β-adducin and PKCδ by substantially inhibiting the expression of β-adducin, spectrin and PKCδ proteins in differentiating keratinocytes. However, extracellular Ca2+ treatment blocked the inhibitory effects of the β-adducin siRNA. Ca2+ also prevented the significant down-regulation of two differentiation markers involucrin and K1/10 and the distinct up-regulation of proliferation marker K14 in β-adducin siRNA transfected keratinocytes. In addition, β-adducin knockdown resulted in a substantial reduction of epidermal growth factor receptor (EGFR), cadherin and β-catenin and enhanced phosphorylation of EGFR on tyrosine 1173 and Ca2+ prevented these changes. Furthermore, Ca2+ blocked the inhibitory effects of β-adducin siRNA on the expression of calmodulin, phosphorylated-calmodulin (P-CaM((Tyr138))) and myristoylated alanine-rich C-kinase substrate (MARCKS) in keratinocytes. Co-immunoprecipitation studies further revealed that calmodulin, not MARCKS, strongly interacted with EGFR, cadherin and β-catenin. Our data suggest that Ca2+ plays an important role in regulating the expression and function of β-adducin to sustain normal organization of the spectrin-based cytoskeleton and the differentiation properties in keratinocytes through the calmodulin/EGFR/cadherin signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Excitation-transcription coupling via calcium/calmodulin-dependent protein kinase/ERK1/2 signaling mediates the coordinate induction of VGLUT2 and Narp triggered by a prolonged increase in glutamatergic synaptic activity.

PubMed

Doyle, Sukhjeevan; Pyndiah, Slovénie; De Gois, Stéphanie; Erickson, Jeffrey D

2010-05-07

Homeostatic scaling of glutamatergic and GABAergic transmission is triggered by prolonged alterations in synaptic neuronal activity. We have previously described a presynaptic mechanism for synaptic homeostasis and plasticity that involves scaling the level of vesicular glutamate (VGLUT1) and gamma-aminobutyric acid (GABA) (VGAT) transporter biosynthesis. These molecular determinants of vesicle filling and quantal size are regulated by neuronal activity in an opposite manner and bi-directionally. Here, we report that a striking induction of VGLUT2 mRNA and synaptic protein is triggered by a prolonged increase in glutamatergic synaptic activity in mature neocortical neuronal networks in vitro together with two determinants of inhibitory synaptic strength, the neuronal activity-regulated pentraxin (Narp), and glutamate decarboxylase (GAD65). Activity-dependent induction of VGLUT2 and Narp exhibits a similar intermediate-early gene response that is blocked by actinomycin D and tetrodotoxin, by inhibitors of ionotropic glutamate receptors and L-type voltage-gated calcium channels, and is dependent on downstream signaling via calmodulin, calcium/calmodulin-dependent protein kinase (CaMK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The co-induction of VGLUT2 and Narp triggered by prolonged gamma-aminobutyric acid type A receptor blockade is independent of brain-derived nerve growth factor and TrkB receptor signaling. VGLUT2 protein induction occurs on a subset of cortically derived synaptic vesicles in excitatory synapses on somata and dendritic processes of multipolar GABAergic interneurons, recognized sites for the clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptors by Narp. We propose that VGLUT2 and Narp induction by excitation-transcription coupling leads to increased glutamatergic transmission at synapses on GABAergic inhibitory feedback neurons as part of a coordinated program of Ca(2+)-signal transcription involved in mechanisms of homeostatic plasticity after prolonged hyperactivity.

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion.

PubMed

Romero, Damian G; Zhou, Ming Yi; Yanes, Licy L; Plonczynski, Maria W; Washington, Tanganika R; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P

2007-08-01

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of MaCaM-Binding Proteins.

PubMed

Jiang, Guoxiang; Wu, Fuwang; Li, Zhiwei; Li, Taotao; Gupta, Vijai Kumar; Duan, Xuewu; Jiang, Yueming

2018-06-01

Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit.

Aspergillus pragensis sp. nov. discovered during molecular reidentification of clinical isolates belonging to Aspergillus section Candidi.

PubMed

Hubka, Vit; Lyskova, Pavlina; Frisvad, Jens C; Peterson, Stephen W; Skorepova, Magdalena; Kolarik, Miroslav

2014-08-01

The identity of nine clinical isolates recovered from Czech patients and presumptively identified as Aspergillus sp. section Candidi based on colony morphology was revised using sequences of β-tubulin, calmodulin gene sequence, and internal transcribed spacer rDNA. Six isolates were from suspected and proven onychomycosis, one from otitis externa, and two associated with probable invasive aspergillosis. The results showed that one Aspergillus candidus isolate was the cause of otitis externa, and both isolates obtained from sputa of patients with probable invasive aspergillosis were reidentified as A. carneus (sect. Terrei) and A. flavus (sect. Flavi). Three isolates from nail scrapings were identified as A. tritici, a verified agent of nondermatophyte onychomycosis. One isolate from toenail was determined to be A. candidus and the two isolates belonged to a hitherto undescribed species, Aspergillus pragensis sp. nov. This species is well supported by phylogenetic analysis based on β-tubulin and calmodulin gene and is distinguishable from other members of sect. Candidi by red-brown reverse on malt extract agar, slow growth on Czapek-Dox agar and inability to grow at 37°C. A secondary metabolite analysis was also provided with comparison of metabolite spectrum to other species. Section Candidi now encompasses five species for which a dichotomous key based on colony characteristics is provided. All clinical isolates were tested for susceptibilities to selected antifungal agents using the Etest and disc diffusion method. Overall sect. Candidi members are highly susceptible to common antifungals. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

PubMed Central

Gaines, Peter; Lamoureux, James; Marisetty, Anantha; Chi, Jeffrey; Berliner, Nancy

2008-01-01

Objective The function of neutrophils as primary mediators of innate immunity depends on the activity of granule proteins and critical components of the NADPH oxidase complex. Expression of their cognate genes is regulated during neutrophil differentiation by a complex network of intracellular signaling pathways. In this study we have investigated the role of two members of the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade, CaMKI-like kinase (CKLiK) and CaMKKα, in regulating neutrophil differentiation and functional activation. Materials and Methods Mouse myeloid cell lines were used to examine the expression of a CaMK cascade in developing neutrophils and to examine the effects of constitutive activation versus inhibition of CaMKs on neutrophil maturation. Results Expression of CaMKKα was shown to increase during neutrophil differentiation in multiple cell lines, whereas expression of CKLiK increased as multipotent progenitors committed to promyelocytes but then decreased as cells differentiated into mature neutrophils. Expression of constitutively active CKLiKs did not affect morphologic maturation, but caused dramatic decreases in both respiratory burst responses and chemotaxis. This loss of neutrophil function was accompanied by reduced secondary granule and gp91phox gene expression. The CaMK inhibitor KN93 attenuated cytokine-stimulated proliferative responses in promyelocytic cell lines, and inhibited the respiratory burst. Similar data were observed with the CaMKKα inhibitor, STO-609. Conclusions Overactivation of a cascade of CaMKs inhibits neutrophil maturation, suggesting that these kinases play an antagonistic role during neutrophil differentiation, but at least one CaMK is required for myeloid cell expansion and functional activation. PMID:18400360

Effect of inhibitors of auxin transport and of calmodulin on a gravisensing-dependent current in maize roots

NASA Technical Reports Server (NTRS)

Bjorkman, T.; Leopold, A. C.

1987-01-01

Some characteristics of the gravity sensing mechanism in maize root caps were investigated using a bioelectric current as an indicator of gravity sensing. This technique involves the measurement of a change in the current density which arises at the columella region coincidently with the presentation time. Two inhibitors of auxin transport, triiodobenzoic acid and naphthylphthalamic acid, blocked gravitropic curvature but not the change in current density. Two inhibitors of calmodulin activity, compound 48/80 and calmidazolium, blocked both curvature and gravity-induced current. The results suggest that auxin transport is not a component of gravity sensing in the root cap. By contrast, the results suggest that calmodulin plays an intrinsic role in gravity sensing.

Overexpression of CsCaM3 Improves High Temperature Tolerance in Cucumber

PubMed Central

Yu, Bingwei; Yan, Shuangshuang; Zhou, Huoyan; Dong, Riyue; Lei, Jianjun; Chen, Changming; Cao, Bihao

2018-01-01

High temperature (HT) stress affects the growth and production of cucumbers, but genetic resources with high heat tolerance are very scarce in this crop. Calmodulin (CaM) has been confirmed to be related to the regulation of HT stress resistance in plants. CsCaM3, a CaM gene, was isolated from cucumber inbred line “02-8.” Its expression was characterized in the present study. CsCaM3 transcripts differed among the organs and tissues of cucumber plants and could be induced by HTs or abscisic acid, but not by salicylic acid. CsCaM3 transcripts exhibited subcellular localization to the cytoplasm and nuclei of cells. Overexpression of CsCaM3 in cucumber plants has the potential to improve their heat tolerance and protect against oxidative damage and photosynthesis system damage by regulating the expression of HT-responsive genes in plants, including chlorophyll catabolism-related genes under HT stress. Taken together, our results provide useful insights into stress tolerance in cucumber. PMID:29946334

Calmodulin shuttling mediates cytonuclear signaling to trigger experience-dependent transcription and memory.

PubMed

Cohen, Samuel M; Suutari, Benjamin; He, Xingzhi; Wang, Yang; Sanchez, Sandrine; Tirko, Natasha N; Mandelberg, Nataniel J; Mullins, Caitlin; Zhou, Guangjun; Wang, Shuqi; Kats, Ilona; Salah, Alejandro; Tsien, Richard W; Ma, Huan

2018-06-22

Learning and memory depend on neuronal plasticity originating at the synapse and requiring nuclear gene expression to persist. However, how synapse-to-nucleus communication supports long-term plasticity and behavior has remained elusive. Among cytonuclear signaling proteins, γCaMKII stands out in its ability to rapidly shuttle Ca 2+ /CaM to the nucleus and thus activate CREB-dependent transcription. Here we show that elimination of γCaMKII prevents activity-dependent expression of key genes (BDNF, c-Fos, Arc), inhibits persistent synaptic strengthening, and impairs spatial memory in vivo. Deletion of γCaMKII in adult excitatory neurons exerts similar effects. A point mutation in γCaMKII, previously uncovered in a case of intellectual disability, selectively disrupts CaM sequestration and CaM shuttling. Remarkably, this mutation is sufficient to disrupt gene expression and spatial learning in vivo. Thus, this specific form of cytonuclear signaling plays a key role in learning and memory and contributes to neuropsychiatric disease.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

NASA Technical Reports Server (NTRS)

Wang, W.; Takezawa, D.; Poovaiah, B. W.

1996-01-01

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

Anterior-posterior regionalized gene expression in the Ciona notochord

PubMed Central

Veeman, Michael

2014-01-01

Background In the simple ascidian chordate Ciona the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape and behavior vary consistently along the anterior-posterior (AP) axis. Results Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL and TGF-β expression are evident even before the notochord cells have intercalated into a single-file column. Conclusions We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. PMID:24288133

Anterior-posterior regionalized gene expression in the Ciona notochord.

PubMed

Reeves, Wendy; Thayer, Rachel; Veeman, Michael

2014-04-01

In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior-posterior (AP) axis. Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF-β expression are evident even before the notochord cells have intercalated into a single-file column. We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. Copyright © 2013 Wiley Periodicals, Inc.

Talaromyces rubrifaciens, a new species discovered from heating, ventilation and air conditioning systems in China.

PubMed

Luo, Yi; Lu, Xiaohong; Bi, Wu; Liu, Fan; Gao, Weiwei

2016-01-01

A new Talaromyces species, T. rubrifaciens, was isolated from supply air outlets of heating, ventilation and air conditioning (HVAC) systems in three kinds of public building in Beijing and Nanjing, China. Morphologically it exhibits many characters of section Trachyspermi but is distinguished from other species of this section by restricted growth and broad and strictly biverticillate conidiophores. Phylogenetic analyses based on the internal transcribed spacer rDNA (ITS), β-tubulin (BenA), calmodulin (CaM) and RNA polymerase second largest subunit (RPB2) genes reveal that T. rubrifaciens is a distinct species in section Trachyspermi. © 2016 by The Mycological Society of America.

The mycoheterotrophic symbiosis between orchids and mycorrhizal fungi possesses major components shared with mutualistic plant-mycorrhizal symbioses.

PubMed

Miura, Chihiro; Yamaguchi, Katsushi; Miyahara, Ryohei; Yamamoto, Tatsuki; Fuji, Masako; Yagame, Takahiro; Imaizumi-Anraku, Haruko; Yamato, Masahide; Shigenobu, Shuji; Kaminaka, Hironori

2018-04-12

Achlorophylous and early developmental stages of chorolophylous orchids are highly dependent on carbon and other nutrients provided by mycorrhizal fungi, in a nutritional mode termed mycoheterotrophy. Previous findings have implied that some common properties at least partially underlie the mycorrhizal symbioses of mycoheterotrophic orchids and that of autotrophic arbuscular mycorrhizal (AM) plants; however, information about the molecular mechanisms of the relationship between orchids and their mycorrhizal fungi is limited. In this study, we characterized the molecular basis of an orchid-mycorrhizal (OM) symbiosis by analyzing the transcriptome of Bletilla striata at an early developmental stage associated with the mycorrhizal fungus Tulasnella sp. The essential components required for the establishment of mutual symbioses with AM fungi and/or rhizobia in most terrestrial plants were identified from B. striata gene set. A cross-species gene complementation analysis showed one of the component genes, calcium and calmodulin-dependent protein kinase gene CCaMK in B. striata, retains functional characteristics of that in AM plants. The expression analysis revealed the activation of homologs of AM-related genes during the OM symbiosis. Our results suggest that orchids possess, at least partly, the molecular mechanisms common to AM plants.

Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28)

USDA-ARS?s Scientific Manuscript database

Plant calcium (Ca2+) dependent protein kinases (CPKs) are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a Calmodulin-like domain (CLD) via an autoinhibitory junction (J) and represent the primary Ca2+-dependent protein kinase activities in plant systems. While regulation...

Chronic ethanol administration inhibits calmodulin-dependent Ca++ uptake in synaptosomal membranes.

PubMed

Ross, D H

1986-06-01

Chronic ethanol administration inhibits ATP-dependent Ca++ uptake in a preparation of synaptic membranes prepared from mice following 1, 4 and 7 days of ethanol exposure in a liquid diet. Addition of calmodulin (2.5 micrograms) to membranes from mice receiving the control diet produced a slight stimulation of ATP dependent Ca++ uptake. Membranes from ETOH treated mice exhibited reduced capacity to take up Ca++ in ATP-dependent fashion. When calmodulin was added to membranes isolated from mice receiving ETOH on Days 1, 4 and 7 ATP-dependent Ca++ uptake was significantly stimulated (p less than 0.01) compared to (1) ETOH treated membranes in absence of calmodulin, and (2) control membranes. Behavioral tolerance as estimated by bar holding technique was found to be 25, 65 and 91 percent complete for Days 1, 4 and 7 respectively. These studies demonstrate that continued exposure of mice to ethanol via consumption of an ethanol containing liquid diet inhibits one of the mechanisms involving the cytosolic buffering of intracellular Ca++ in nerve terminals. This biochemical effect seen in parallel with the development of tolerance to ethanol impairment of bar holding suggests that increased cytosolic Ca++ may aid in central nervous system adaptation to ethanol.

NMR structural study of the intracellular loop 3 of the serotonin 5-HT(1A) receptor and its interaction with calmodulin.

PubMed

Chen, Angela Shuyi; Kim, Young Mee; Gayen, Shovanlal; Huang, Qiwei; Raida, Manfred; Kang, Congbao

2011-09-01

The serotonin (5-HT(1A)) receptor, a G-protein-coupled receptor (GPCR), plays important roles in serotonergic signaling in the central nervous system. The third intracellular loop (ICL3) of the 5-HT(1A) receptor has been shown to be important for the regulation of this receptor through interactions with proteins such as G-proteins and calmodulin. In this study, the ICL3 of 5-HT(1A) receptor was expressed in E. coli and purified. Gel filtration and mass spectrometry were used to confirm the molecular weight of the purified ICL3. Secondary structure analysis using circular dichroism (CD) demonstrated the presence of α-helical structures. Backbone assignment of ICL3 was achieved using three-dimensional experiments. A chemical shift index and Talos+ analysis showed that residues E326 to R339 form α-helical structure. Residues G256 to S269 of ICL3 were shown to be a novel region that has a molecular interaction with calmodulin in titration assays. Peptide derived from the ICL3 containing residues from G256 to S269 also showed molecular interaction with calmodulin. Copyright © 2011 Elsevier B.V. All rights reserved.

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells.

PubMed

Kurosaki, F; Kaburaki, H; Nishi, A

1994-03-07

Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 microM of free Ca2+, while Ca2+ lower than 0.1 microM did not affect the Ca(2+)-release. The Ca2+ flux across plasma membrane was restored from this Ca(2+)-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca2+ channel.

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase

NASA Astrophysics Data System (ADS)

Wagner, John A.; Cozens, Alison L.; Schulman, Howard; Gruenert, Dieter C.; Stryer, Lubert; Gardner, Phyllis

1991-02-01

CYSTIC fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase1,2 and protein kinase C3,4. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels1-4. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2+-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2+-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca2+ -calmodulin at mossy fiber-CA3 synapses.

PubMed

Andrade-Talavera, Yuniesky; Duque-Feria, Paloma; Negrete-Díaz, José Vicente; Sihra, Talvinder S; Flores, Gonzalo; Rodríguez-Moreno, Antonio

2012-09-01

Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Species concepts in Cercospora: spotting the weeds among the roses

PubMed Central

Groenewald, J.Z.; Nakashima, C.; Nishikawa, J.; Shin, H.-D.; Park, J.-H.; Jama, A.N.; Groenewald, M.; Braun, U.; Crous, P.W.

2013-01-01

The genus Cercospora contains numerous important plant pathogenic fungi from a diverse range of hosts. Most species of Cercospora are known only from their morphological characters in vivo. Although the genus contains more than 5 000 names, very few cultures and associated DNA sequence data are available. In this study, 360 Cercospora isolates, obtained from 161 host species, 49 host families and 39 countries, were used to compile a molecular phylogeny. Partial sequences were derived from the internal transcribed spacer regions and intervening 5.8S nrRNA, actin, calmodulin, histone H3 and translation elongation factor 1-alpha genes. The resulting phylogenetic clades were evaluated for application of existing species names and five novel species are introduced. Eleven species are epi-, lecto- or neotypified in this study. Although existing species names were available for several clades, it was not always possible to apply North American or European names to African or Asian strains and vice versa. Some species were found to be limited to a specific host genus, whereas others were isolated from a wide host range. No single locus was found to be the ideal DNA barcode gene for the genus, and species identification needs to be based on a combination of gene loci and morphological characters. Additional primers were developed to supplement those previously published for amplification of the loci used in this study. Taxonomic novelties: New species - Cercospora coniogrammes Crous & R.G. Shivas, Cercospora delaireae C. Nakash., Crous, U. Braun & H.D. Shin, Cercospora euphorbiae-sieboldianae C. Nakash., Crous, U. Braun & H.D. Shin, Cercospora pileicola C. Nakash., Crous, U. Braun & H.D. Shin, Cercospora vignigena C. Nakash., Crous, U. Braun & H.D. Shin. Typifications: epitypifications - Cercospora alchemillicola U. Braun & C.F. Hill, Cercospora althaeina Sacc., Cercospora armoraciae Sacc., Cercospora corchori Sawada, Cercospora mercurialis Pass., Cercospora olivascens Sacc., Cercospora violae Sacc.; neotypifications - Cercospora fagopyri N. Nakata & S. Takim., Cercospora sojina Hara. PMID:24014899

Egr-1 antisense oligodeoxynucleotide administration into the olfactory bulb impairs olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx.

PubMed

Ganesh, Ambigapathy; Bogdanowicz, Wieslaw; Balamurugan, Krishnaswamy; Ragu Varman, Durairaj; Rajan, Koilmani Emmanuvel

2012-08-30

Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We examined two important examples of these, calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95, in regard to the functional role of early growth response gene-1 (egr-1) in regulation of olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx (family Pteropodidae). To test whether activation of egr-1 in the olfactory bulb (OB) is required for olfactory memory of these bats, bilaterally canulated individuals were infused with antisense (AS) or non-sense (NS)-oligodeoxynucleotides (ODN) of egr-1, or with phosphate buffer saline (PBS), 2h before the olfactory training. Our results showed that behavioral training significantly up-regulates immediate early gene (IEG) EGR-1 and key synaptic proteins Synaptotagmin-1(SYT-1), CaMKII and PSD-95, and phosphorylation of CaMKII in the OB at the protein level per se. Subsequently, we observed that egr-1 antisense-ODN infusion in the OB impaired olfactory memory and down regulates the expression of CaMKII and PSD-95, and the phosphorylation of CaMKII but not SYT-1. In contrast, NS-ODN or PBS had no effect on the expression of the PSDs CaMKII or PSD-95, or on the phosphorylation of CaMKII. When the egr-1 NS-ODN was infused in the OB after training for the novel odor there was no effect on olfactory memory. These findings suggest that egr-1 control the activation of CaMKII and PSD-95 during the process of olfactory memory formation. Copyright © 2012 Elsevier B.V. All rights reserved.

Food anticipatory activity on a calorie-restricted diet is independent of Sirt1.

PubMed

Assali, Dina R; Hsu, Cynthia T; Gunapala, Keith M; Aguayo, Antonio; McBurney, Michael; Steele, Andrew D

2018-01-01

A number of studies have demonstrated that the Sirtuin family member, Sirt1, is a key integrator of growth, metabolism, and lifespan. Sirt1 directly interacts with and deacetylates key regulators of the circadian clock, positioning it to be an important link between feeding and circadian rhythms. In fact, one study suggests that Sirt1 is necessary for behavioral anticipation of limited daily food availability, a circadian process termed food anticipatory activity (FAA). In their study, mice overexpressing Sirt1 had enhanced FAA, while mice lacking Sirt1 had little to no FAA. Based on the supposition that Sirt1 was indeed required for FAA, we sought to use Sirt1 deletion to map the neural circuitry responsible for FAA. We began by inactivating Sirt1 using the cell-type specific Cre-driver lines proopiomelanocortin, but after observing no effect on body weight loss or FAA we then moved on to more broadly neuronal Cre drivers Ca2+/calmodulin-dependent protein kinase II and nestin. As neither of these neuronal deletions of Sirt1 had impaired FAA, we then tested 1) a broad postnatal tamoxifen-inducible deletion, 2) a complete, developmental knockout of Sirt1, and 3) a gene replacement, catalytically inactive, form of Sirt1; but all of these mice had FAA similar to controls. Therefore, our findings suggest that FAA is completely independent of Sirt1.

Calcium signals act through histone deacetylase to mediate pronephric kidney morphogenesis.

PubMed

Rothschild, Sarah C; Lee, Hunter J; Ingram, Sarah R; Mohammadi, Daniel K; Walsh, Gregory S; Tombes, Robert M

2018-06-01

Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca 2+ -conducting TRP channel enriched in ciliated cells and gated by extracellular signals, is necessary to activate the multifunctional Ca 2+/ calmodulin-dependent protein kinase type 2 (CaMK-II), enabling kidney morphogenesis and cilia stability. In this study, antisense morpholino oligonucleotides and pharmacological compounds were employed to investigate the roles of class II HDAC family members (HDAC 4, 5, and 6) in Zebrafish kidney development. While all three class II HDAC genes were expressed throughout the embryo during early development, HDAC5-morphant embryos exhibited anterior cysts and destabilized cloacal cilia, similar to PKD2 and CaMK-II morphants. In contrast, HDAC4-morphant embryos exhibited elongated cloacal cilia and lacked anterior kidney defects. Suppression of HDAC4 partially reversed the cilia shortening and anterior convolution defects caused by CaMK-II deficiency, whereas HDAC5 loss exacerbated these defects. EGFP-HDAC4, but not EGFP-HDAC5, translocated into the nucleus upon CaMK-II suppression in pronephric kidney cells. These results support a model by which activated CaMK-II sequesters HDAC4 in the cytosol to enable primary cilia formation and kidney morphogenesis. Developmental Dynamics 247:807-817, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells.

PubMed

Luby-Phelps, K; Lanni, F; Taylor, D L

1985-10-01

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

PubMed Central

1985-01-01

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole- phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm. PMID:4044638

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

NASA Technical Reports Server (NTRS)

Roux, S. J.

1990-01-01

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

Calcium ion binding properties of Medicago truncatula calcium/calmodulin-dependent protein kinase.

PubMed

Swainsbury, David J K; Zhou, Liang; Oldroyd, Giles E D; Bornemann, Stephen

2012-09-04

A calcium/calmodulin-dependent protein kinase (CCaMK) is essential in the interpretation of calcium oscillations in plant root cells for the establishment of symbiotic relationships with rhizobia and mycorrhizal fungi. Some of its properties have been studied in detail, but its calcium ion binding properties and subsequent conformational change have not. A biophysical approach was taken with constructs comprising either the visinin-like domain of Medicago truncatula CCaMK, which contains EF-hand motifs, or this domain together with the autoinhibitory domain. The visinin-like domain binds three calcium ions, leading to a conformational change involving the exposure of hydrophobic surfaces and a change in tertiary but not net secondary or quaternary structure. The affinity for calcium ions of visinin-like domain EF-hands 1 and 2 (K(d) = 200 ± 50 nM) was appropriate for the interpretation of calcium oscillations (~125-850 nM), while that of EF-hand 3 (K(d) ≤ 20 nM) implied occupancy at basal calcium ion levels. Calcium dissociation rate constants were determined for the visinin-like domain of CCaMK, M. truncatula calmodulin 1, and the complex between these two proteins (the slowest of which was 0.123 ± 0.002 s(-1)), suggesting the corresponding calcium association rate constants were at or near the diffusion-limited rate. In addition, the dissociation of calmodulin from the protein complex was shown to be on the same time scale as the dissociation of calcium ions. These observations suggest that the formation and dissociation of the complex between calmodulin and CCaMK would substantially mirror calcium oscillations, which typically have a 90 s periodicity.

Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation.

PubMed

Yan, Jin; Leal, Karina; Magupalli, Venkat G; Nanou, Evanthia; Martinez, Gilbert Q; Scheuer, Todd; Catterall, William A

2014-11-01

Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.

Ryanodine receptor phosphorylation by calcium/calmodulin-dependent protein kinase II promotes life-threatening ventricular arrhythmias in mice with heart failure.

PubMed

van Oort, Ralph J; McCauley, Mark D; Dixit, Sayali S; Pereira, Laetitia; Yang, Yi; Respress, Jonathan L; Wang, Qiongling; De Almeida, Angela C; Skapura, Darlene G; Anderson, Mark E; Bers, Donald M; Wehrens, Xander H T

2010-12-21

approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

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

NASA Technical Reports Server (NTRS)

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.

Genome-Wide Analyses of Calcium Sensors Reveal Their Involvement in Drought Stress Response and Storage Roots Deterioration after Harvest in Cassava.

PubMed

Hu, Wei; Yan, Yan; Tie, Weiwei; Ding, Zehong; Wu, Chunlai; Ding, Xupo; Wang, Wenquan; Xia, Zhiqiang; Guo, Jianchun; Peng, Ming

2018-04-19

Calcium (Ca 2+ ) plays a crucial role in plant development and responses to environmental stimuli. Currently, calmodulins (CaMs), calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs), such as Ca 2+ sensors, are not well understood in cassava ( Manihot esculenta Crantz), an important tropical crop. In the present study, 8 CaMs, 48 CMLs, and 9 CBLs were genome-wide identified in cassava, which were divided into two, four, and four groups, respectively, based on evolutionary relationship, protein motif, and gene structure analyses. Transcriptomic analysis revealed the expression diversity of cassava CaMs-CMLs-CBLs in distinct tissues and in response to drought stress in different genotypes. Generally, cassava CaMs-CMLs-CBLs showed different expression profiles between cultivated varieties (Arg7 and SC124) and wild ancestor (W14) after drought treatment. In addition, numerous CaMs-CMLs-CBLs were significantly upregulated at 6 h, 12 h, and 48 h after harvest, suggesting their possible role during storage roots (SR) deterioration. Further interaction network and co-expression analyses suggested that a CBL-mediated interaction network was widely involved in SR deterioration. Taken together, this study provides new insights into CaMs-CMLs-CBLs-mediated drought adaption and SR deterioration at the transcription level in cassava, and identifies some candidates for the genetic improvement of cassava.

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin

PubMed Central

Wafer, Lucas N; Tzul, Franco O; Pandharipande, Pranav P; McCallum, Scott A; Makhatadze, George I

2014-01-01

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins. PMID:24947426

Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Qin, E-mail: yang@crystal.harvard.edu; Brüschweiler, Sven; Chou, James J., E-mail: yang@crystal.harvard.edu

2013-12-24

The N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1 was crystallized in the presence of Ca{sup 2+}. X-ray diffraction data were collected to 2.9 Å resolution from crystals which belonged to space group P6{sub 2}22.

Cyclic Nucleotide-Gated Channels, Calmodulin, Adenylyl Cyclase, and Calcium/Calmodulin-Dependent Protein Kinase II Are Required for Late, but Not Early, Long-Term Memory Formation in the Honeybee

ERIC Educational Resources Information Center

Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

2014-01-01

Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee "Apis mellifera," olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM)…

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

PubMed

Saito, Kazuki; Nakato, Mamiko; Mizuguchi, Takaaki; Wada, Shinji; Uchimura, Hiromasa; Kataoka, Hiroshi; Yokoyama, Shigeyuki; Hirota, Hiroshi; Kiso, Yoshiaki

2014-03-01

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, β-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 μM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of calcineurin by phosphotidylserine containing vesicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Politino, M.; King, M.M.

1986-05-01

Calcineurin (CaN) is a Ca/sup 2 +/- and calmodulin-regulated phosphatase. Recent findings suggested an association of CaN with biological membranes and prompted the present investigation into the interactions of the phosphatase with phospholipids in vitro. In the absence of calmodulin, sonicated preparations of phosphatidylserine (PS) provided a five-fold activation of the Ni- and Mn-supported activities of CaN towards (/sup 32/P) histone Hl; activation in the presence of calmodulin was much less pronounced. Half-maximal activation in the absence of calmodulin required approximately 0.1 mg/ml of PS. Activation of CaN was also observed with mixed vesicles of phosphatidylcholine (PC) containing 20% PSmore » but not with PC alone, or with phosphatidylethanolamine (PE). Molecular sieve chromatography on Ultrogel AcA 34 provided further evidence that CaN associates with phospholipid vesicles composed of PS, or PC containing 20% PS, but not with vesicles of PC or PE. Complete association with medium sized vesicles of PS and PC/PS required Ca/sup 2 +/ ions; in the absence of the metal ion at least 60% of the enzyme failed to interact with the lipids while the remainder preferentially migrated with larger vesicles. These results suggest a role for Ca/sup 2 +/ in regulating CaN's interaction with phospholipids.« less

Role of calcium activated kinases and phosphatases in heat shock factor-1 activation.

PubMed

Soncin, F; Asea, A; Zhang, X; Stevenson, M A; Calderwood, S K

2000-12-01

HSF-1 is regulated at multiple molecular levels through intra- and intermolecular protein-protein interactions as well as by post-translational modification through phosphorylation. We have found that elevating intracellular calcium ion levels by exposure to the ionophore A23187 or thapsigargin inhibits the conversion of HSF-1 from a latent cytoplasmic form to its nuclear/DNA binding form. To examine a role for calcium/calmodulin regulated enzymes in this process, we examined the ability of specific inhibitors to abrogate the effects of calcium elevation. While the inhibitor of calmodulin dependent kinase II, KCN62 enhanced activation of HSF-1 during heat shock, it failed to block the inhibitory effects of calcium increase. By contrast, the immunosuppresant drugs cyclosporin A and FK506 abolished the effects of calcium elevation on HSF-1 activation. As the biological effects of the drugs are effected through inhibition of the calcium/calmodulin regulated phosphatase calcineurin, this suggests a role for calcineurin in antagonizing HSF-1 activity. The experiments suggest the existence of phosphorylated residue(s) in HSF-1 important in one or more of the processes that lead to activation (trimerization, nuclear localization, DNA binding) and which becomes dephosphorylated due to the activation of a calcium/calmodulin/calcineurin complex.

Platelet interactions in thrombosis.

PubMed

Andrews, Robert K; Gardiner, Elizabeth E; Shen, Yang; Berndt, Michael C

2004-01-01

Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca2+, dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and 'inside-out' activation of the integrin, alphaIIbbeta3 (GPIIb-llla), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical co-association of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease.

Transcriptome analysis of genes involved in defense against alkaline stress in roots of wild jujube (Ziziphus acidojujuba)

PubMed Central

Tian, Shan; Wang, Bei; Zhao, Xusheng

2017-01-01

Wild jujube (Ziziphus acidojujuba Mill.) is highly tolerant to alkaline, saline and drought stress; however, no studies have performed transcriptome profiling to study the response of wild jujube to these and other abiotic stresses. In this study, we examined the tolerance of wild jujube to NaHCO3-NaOH solution and analyzed gene expression profiles in response to alkaline stress. Physiological experiments revealed that H2O2 content in leaves increased significantly and root activity decreased quickly during alkaline of pH 9.5 treatment. For transcriptome analysis, wild jujube plants grown hydroponically were treated with NaHCO3-NaOH solution for 0, 1, and 12 h and six transcriptomes from roots were built. In total, 32,758 genes were generated, and 3,604 differentially expressed genes (DEGs) were identified. After 1 h, 853 genes showed significantly different expression between control and treated plants; after 12 h, expression of 2,856 genes was significantly different. The expression pattern of nine genes was validated by quantitative real-time PCR. After gene annotation and gene ontology enrichment analysis, the genes encoding transcriptional factors, serine/threonine-protein kinases, heat shock proteins, cysteine-like kinases, calmodulin-like proteins, and reactive oxygen species (ROS) scavengers were found to be closely involved in alkaline stress response. These results will provide useful insights for elucidating the mechanisms underlying alkaline tolerance in wild jujube. PMID:28976994

Gene family size conservation is a good indicator of evolutionary rates.

PubMed

Chen, Feng-Chi; Chen, Chiuan-Jung; Li, Wen-Hsiung; Chuang, Trees-Juen

2010-08-01

The evolution of duplicate genes has been a topic of broad interest. Here, we propose that the conservation of gene family size is a good indicator of the rate of sequence evolution and some other biological properties. By comparing the human-chimpanzee-macaque orthologous gene families with and without family size conservation, we demonstrate that genes with family size conservation evolve more slowly than those without family size conservation. Our results further demonstrate that both family expansion and contraction events may accelerate gene evolution, resulting in elevated evolutionary rates in the genes without family size conservation. In addition, we show that the duplicate genes with family size conservation evolve significantly more slowly than those without family size conservation. Interestingly, the median evolutionary rate of singletons falls in between those of the above two types of duplicate gene families. Our results thus suggest that the controversy on whether duplicate genes evolve more slowly than singletons can be resolved when family size conservation is taken into consideration. Furthermore, we also observe that duplicate genes with family size conservation have the highest level of gene expression/expression breadth, the highest proportion of essential genes, and the lowest gene compactness, followed by singletons and then by duplicate genes without family size conservation. Such a trend accords well with our observations of evolutionary rates. Our results thus point to the importance of family size conservation in the evolution of duplicate genes.

Genome-Wide Comparative Gene Family Classification

PubMed Central

Frech, Christian; Chen, Nansheng

2010-01-01

Correct classification of genes into gene families is important for understanding gene function and evolution. Although gene families of many species have been resolved both computationally and experimentally with high accuracy, gene family classification in most newly sequenced genomes has not been done with the same high standard. This project has been designed to develop a strategy to effectively and accurately classify gene families across genomes. We first examine and compare the performance of computer programs developed for automated gene family classification. We demonstrate that some programs, including the hierarchical average-linkage clustering algorithm MC-UPGMA and the popular Markov clustering algorithm TRIBE-MCL, can reconstruct manual curation of gene families accurately. However, their performance is highly sensitive to parameter setting, i.e. different gene families require different program parameters for correct resolution. To circumvent the problem of parameterization, we have developed a comparative strategy for gene family classification. This strategy takes advantage of existing curated gene families of reference species to find suitable parameters for classifying genes in related genomes. To demonstrate the effectiveness of this novel strategy, we use TRIBE-MCL to classify chemosensory and ABC transporter gene families in C. elegans and its four sister species. We conclude that fully automated programs can establish biologically accurate gene families if parameterized accordingly. Comparative gene family classification finds optimal parameters automatically, thus allowing rapid insights into gene families of newly sequenced species. PMID:20976221

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

PubMed Central

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca2+

PubMed Central

Sakato, Miho; Sakakibara, Hitoshi

2007-01-01

We have previously shown that Ca2+ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the β and γ heavy chains (HCs). The γ HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca2+ with KCa = 3 × 10−5 M in vitro, suggesting it may act as a Ca2+ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and γ HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the γ heavy chain. In vitro experiments indicate that LC4 undergoes a Ca2+-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca2+. The sedimentation profile of the γ HC subunit changed subtly upon Ca2+ addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated γ subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca2+ addition. We propose that Ca2+-dependent conformational change of LC4 has a direct effect on the stem domain of the γ HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm. PMID:17634291

Phlebotomy-induced anemia alters hippocampal neurochemistry in neonatal mice

PubMed Central

Wallin, Diana J.; Tkac, Ivan; Stucker, Sara; Ennis, Kathleen M.; Sola-Visner, Martha; Rao, Raghavendra; Georgieff, Michael K.

2015-01-01

Background Phlebotomy-induced anemia (PIA) is common in preterm infants. The hippocampus undergoes rapid differentiation during late fetal/early neonatal life and relies on adequate oxygen and iron to support oxidative metabolism necessary for development. Anemia shortchanges these two critical substrates, potentially altering hippocampal development and function. Methods PIA (hematocrit <25%) was induced in neonatal mice pups from postnatal day (P)3 to P14. Neurochemical concentrations in the hippocampus were determined using in vivo 1H NMR spectroscopy at 9.4T and compared with control animals at P14. Gene expression was assessed using qRT-PCR. Results PIA decreased brain iron concentration, increased hippocampal lactate and creatine concentrations, and decreased phosphoethanolamine (PE) concentration and the phosphocreatine/creatine ratio. Hippocampal transferrin receptor (Tfrc) gene expression was increased, while the expression of calcium/calmodulin-dependent protein kinase type II alpha (CamKIIα) was decreased in PIA mice. Conclusion This clinically relevant model of neonatal anemia alters hippocampal energy and phospholipid metabolism and gene expression during a critical developmental period. Low target hematocrits for preterm neonates in the NICU may have potential adverse neural implications. PMID:25734245

Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gangopadhyay, Jaya Pal, E-mail: jaya@bbri.org; Ikemoto, Noriaki; Department of Neurology, Harvard Medical School, Boston, MA 02115

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca{sup 2+} leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in themore » nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca{sup 2+}/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca{sup 2+} transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca{sup 2+} events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.« less

Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Kalari Satish; Ravi Kumar, B.; Siddavattam, Dayananda

2006-07-07

In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtainedmore » from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory {beta}-subunit of calcineurin (Ca{sup 2+}-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.« less

Population resequencing reveals candidate genes associated with salinity adaptation of the Pacific oyster Crassostrea gigas.

PubMed

She, Zhicai; Li, Li; Meng, Jie; Jia, Zhen; Que, Huayong; Zhang, Guofan

2018-06-06

The Pacific oyster Crassostrea gigas is an important cultivated shellfish. As a euryhaline species, it has evolved adaptive mechanisms responding to the complex and changeable intertidal environment that it inhabits. To investigate the genetic basis of this salinity adaptation mechanism, we conducted a genome-wide association study using phenotypically differentiated populations (hyposalinity and hypersalinity adaptation populations, and control population), and confirmed our results using an independent population, high-resolution melting, and mRNA expression analysis. For the hyposalinity adaptation, we determined 24 genes, including Cg_CLCN7 (chloride channel protein 7) and Cg_AP1 (apoptosis 1 inhibitor), involved in the ion/water channel and transporter mechanisms, free amino acid and reactive oxygen species metabolism, immune responses, and chemical defence. Three SNPs located on these two genes were significantly differentiated between groups, as was Cg_CLCN7. For the hypersalinity adaptation, the biological process for positive regulating the developmental process was enriched. Enriched gene functions were focused on transcriptional regulation, signal transduction, and cell growth and differentiation, including calmodulin (Cg_CaM) and ficolin-2 (Cg_FCN2). These genes and polymorphisms possibly play an important role in oyster hyposalinity and hypersalinity adaptation. They not only further our understanding of salinity adaptation mechanisms but also provide markers for highly adaptable oyster strains suitable for breeding.

Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

2015-12-10

D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. Copyright © 2015 Elsevier B.V. All rights reserved.

Electrophoretic mobility shift in native gels indicates calcium-dependent structural changes of neuronal calcium sensor proteins.

PubMed

Viviano, Jeffrey; Krishnan, Anuradha; Wu, Hao; Venkataraman, Venkat

2016-02-01

In proteins of the neuronal calcium sensor (NCS) family, changes in structure as well as function are brought about by the binding of calcium. In this article, we demonstrate that these structural changes, solely due to calcium binding, can be assessed through electrophoresis in native gels. The results demonstrate that the NCS proteins undergo ligand-dependent conformational changes that are detectable in native gels as a gradual decrease in mobility with increasing calcium but not other tested divalent cations such as magnesium, strontium, and barium. Surprisingly, such a gradual change over the entire tested range is exhibited only by the NCS proteins but not by other tested calcium-binding proteins such as calmodulin and S100B, indicating that the change in mobility may be linked to a unique NCS family feature--the calcium-myristoyl switch. Even within the NCS family, the changes in mobility are characteristic of the protein, indicating that the technique is sensitive to the individual features of the protein. Thus, electrophoretic mobility on native gels provides a simple and elegant method to investigate calcium (small ligand)-induced structural changes at least in the superfamily of NCS proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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+).

Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

PubMed Central

Xie, Jianling; Mikolajek, Halina; Pigott, Craig R.; Hooper, Kelly J.; Mellows, Toby; Moore, Claire E.; Mohammed, Hafeez; Werner, Jörn M.; Thomas, Gareth J.

2015-01-01

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis. PMID:25776553

Rapid and transient stimulation of intracellular reactive oxygen species by melatonin in normal and tumor leukocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Radogna, Flavia; Paternoster, Laura; Istitututo di Chimica Biologica, Universita di Urbino Carlo Bo

Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (< 1 min) and transient (up to 5-6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal or tumor leukocytes; intriguingly, ROS production does not leadmore » to oxidative stress, as shown by absence of protein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROS production is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed to nanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is not mimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulin inhibitor shown to prevent melatonin-calmodulin interaction, also prevents melatonin pro-radical effect, suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROS production.« less

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

PubMed

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.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

PubMed

Zilio, Nicola; Boddy, Michael N

2017-03-01

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins. © 2017 Cold Spring Harbor Laboratory Press.

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects.

PubMed

Eischen-Loges, Maria; Oliveira, Karla M C; Bhavsar, Mit B; Barker, John H; Leppik, Liudmila

2018-01-01

Electrical stimulation (ES) has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES's demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE). In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC) osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1 , Osteopontin , Osterix and Calmodulin . We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days) after discontinuing ES exposure. This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This prolonged positive osteogenic effect, long after discontinuing ES treatment, if incorporated into BTE treatment protocols, could potentially improve outcomes and in doing so help BTE achieve its full therapeutic potential.

Calcium/calmodulin-dependent serine protein kinase (CASK), a protein implicated in mental retardation and autism-spectrum disorders, interacts with T-Brain-1 (TBR1) to control extinction of associative memory in male mice.

PubMed

Huang, Tzyy-Nan; Hsueh, Yi-Ping

2017-01-01

Human genetic studies have indicated that mutations in calcium/calmodulin-dependent serine protein kinase ( CASK ) result in X-linked mental retardation and autism-spectrum disorders. We aimed to establish a mouse model to study how Cask regulates mental ability. Because Cask encodes a multidomain scaffold protein, a possible strategy to dissect how CASK regulates mental ability and cognition is to disrupt specific protein-protein interactions of CASK in vivo and then investigate the impact of individual specific protein interactions. Previous in vitro analyses indicated that a rat CASK T724A mutation reduces the interaction between CASK and T-brain-1 (TBR1) in transfected COS cells. Because TBR1 is critical for glutamate receptor, ionotropic, N -methyl-D-aspartate receptor subunit 2B ( Grin2b ) expression and is a causative gene for autism and intellectual disability, we then generated CASK T740A (corresponding to rat CASK T724A) mutant mice using a gene-targeting approach. Immunoblotting, coimmunoprecipitation, histological methods and behavioural assays (including home cage, open field, auditory and contextual fear conditioning and conditioned taste aversion) were applied to investigate expression of CASK and its related proteins, the protein-protein interactions of CASK, and anatomic and behavioural features of CASK T740A mice. The CASK T740A mutation attenuated the interaction between CASK and TBR1 in the brain. However, CASK T740A mice were generally healthy, without obvious defects in brain morphology. The most dramatic defect among the mutant mice was in extinction of associative memory, though acquisition was normal. The functions of other CASK protein interactions cannot be addressed using CASK T740A mice. Disruption of the CASK and TBR1 interaction impairs extinction, suggesting the involvement of CASK in cognitive flexibility.

Sexy gene conversions: locating gene conversions on the X-chromosome.

PubMed

Lawson, Mark J; Zhang, Liqing

2009-08-01

Gene conversion can have a profound impact on both the short- and long-term evolution of genes and genomes. Here, we examined the gene families that are located on the X-chromosomes of human (Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus) and rat (Rattus norvegicus) for evidence of gene conversion. We identified seven gene families (WD repeat protein family, Ferritin Heavy Chain family, RAS-related Protein RAB-40 family, Diphosphoinositol polyphosphate phosphohydrolase family, Transcription Elongation Factor A family, LDOC1-related family, Zinc Finger Protein ZIC, and GLI family) that show evidence of gene conversion. Through phylogenetic analyses and synteny evidence, we show that gene conversion has played an important role in the evolution of these gene families and that gene conversion has occurred independently in both primates and rodents. Comparing the results with those of two gene conversion prediction programs (GENECONV and Partimatrix), we found that both GENECONV and Partimatrix have very high false negative rates (i.e. failed to predict gene conversions), which leads to many undetected gene conversions. The combination of phylogenetic analyses with physical synteny evidence exhibits high resolution in the detection of gene conversions.

Plasticity in the Rat Prefrontal Cortex: Linking Gene Expression and an Operant Learning with a Computational Theory

PubMed Central

Rapanelli, Maximiliano; Lew, Sergio Eduardo; Frick, Luciana Romina; Zanutto, Bonifacio Silvano

2010-01-01

The plasticity in the medial Prefrontal Cortex (mPFC) of rodents or lateral prefrontal cortex in non human primates (lPFC), plays a key role neural circuits involved in learning and memory. Several genes, like brain-derived neurotrophic factor (BDNF), cAMP response element binding (CREB), Synapsin I, Calcium/calmodulin-dependent protein kinase II (CamKII), activity-regulated cytoskeleton-associated protein (Arc), c-jun and c-fos have been related to plasticity processes. We analysed differential expression of related plasticity genes and immediate early genes in the mPFC of rats during learning an operant conditioning task. Incompletely and completely trained animals were studied because of the distinct events predicted by our computational model at different learning stages. During learning an operant conditioning task, we measured changes in the mRNA levels by Real-Time RT-PCR during learning; expression of these markers associated to plasticity was incremented while learning and such increments began to decline when the task was learned. The plasticity changes in the lPFC during learning predicted by the model matched up with those of the representative gene BDNF. Herein, we showed for the first time that plasticity in the mPFC in rats during learning of an operant conditioning is higher while learning than when the task is learned, using an integrative approach of a computational model and gene expression. PMID:20111591

Cooperative binding mitigates the high-dose hook effect.

PubMed

Roy, Ranjita Dutta; Rosenmund, Christian; Stefan, Melanie I

2017-08-14

The high-dose hook effect (also called prozone effect) refers to the observation that if a multivalent protein acts as a linker between two parts of a protein complex, then increasing the amount of linker protein in the mixture does not always increase the amount of fully formed complex. On the contrary, at a high enough concentration range the amount of fully formed complex actually decreases. It has been observed that allosterically regulated proteins seem less susceptible to this effect. The aim of this study was two-fold: First, to investigate the mathematical basis of how allostery mitigates the prozone effect. And second, to explore the consequences of allostery and the high-dose hook effect using the example of calmodulin, a calcium-sensing protein that regulates the switch between long-term potentiation and long-term depression in neurons. We use a combinatorial model of a "perfect linker protein" (with infinite binding affinity) to mathematically describe the hook effect and its behaviour under allosteric conditions. We show that allosteric regulation does indeed mitigate the high-dose hook effect. We then turn to calmodulin as a real-life example of an allosteric protein. Using kinetic simulations, we show that calmodulin is indeed subject to a hook effect. We also show that this effect is stronger in the presence of the allosteric activator Ca 2+ /calmodulin-dependent kinase II (CaMKII), because it reduces the overall cooperativity of the calcium-calmodulin system. It follows that, surprisingly, there are conditions where increased amounts of allosteric activator actually decrease the activity of a protein. We show that cooperative binding can indeed act as a protective mechanism against the hook effect. This will have implications in vivo where the extent of cooperativity of a protein can be modulated, for instance, by allosteric activators or inhibitors. This can result in counterintuitive effects of decreased activity with increased concentrations of both the allosteric protein itself and its allosteric activators.

Superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel.

PubMed

Kawakami, M; Okabe, E

1998-03-01

The ryanodine receptor Ca2+ channel (RyRC) constitutes the Ca2+-release pathway in sarcoplasmic reticulum (SR) of cardiac muscle. A direct mechanical and a Ca2+-triggered mechanism (Ca2+-induced Ca2+ release) have been proposed to explain the in situ activation of Ca2+ release in cardiac muscle. A variety of chemical oxidants have been shown to activate RyRC; however, the role of modification induced by oxygen-derived free radicals in pathological states of the muscle remains to be elucidated. It has been hypothesized that oxygen-derived free radicals initiate Ca2+-mediated functional changes in or damage to cardiac muscle by acting on the SR and promoting an increase in Ca2+ release. We confirmed that superoxide anion radical (O2-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles; hypoxanthine-xanthine oxidase also decreases Ca2+ free within the intravesicular space of the SR with no effect on Ca2+-ATPase activity. Current fluctuations through single Ca2+-release channels have been monitored after incorporation into planar phospholipid bilayers. We demonstrate that activation of the channel by O2- is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O2--triggered Ca2+ release through the RyRC. For the first time, we show that O2- stimulates Ca2+ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2-. The decreased calmodulin content induced by oxygen-derived free radicals, especially O2-, is a likely mechanism of accumulation of cytosolic Ca2+ (due to increased Ca2+ release from SR) after reperfusion of the ischemic heart.

Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

PubMed

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.

PubMed

Ihling, Christian; Schmidt, Andreas; Kalkhof, Stefan; Schulz, Daniela M; Stingl, Christoph; Mechtler, Karl; Haack, Michael; Beck-Sickinger, Annette G; Cooper, Dermot M F; Sinz, Andrea

2006-08-01

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.

CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo

PubMed Central

Passier, Robert; Zeng, Hong; Frey, Norbert; Naya, Francisco J.; Nicol, Rebekka L.; McKinsey, Timothy A.; Overbeek, Paul; Richardson, James A.; Grant, Stephen R.; Olson, Eric N.

2000-01-01

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca2+-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca2+/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy. PMID:10811847

TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

PubMed

Parvin, Nargis; Carrie, Chris; Pabst, Isabelle; Läßer, Antonia; Laha, Debabrata; Paul, Melanie V; Geigenberger, Peter; Heermann, Ralf; Jung, Kirsten; Vothknecht, Ute C; Chigri, Fatima

2017-04-03

The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Conformational Contribution to Thermodynamics of Binding in Protein-Peptide Complexes through Microscopic Simulation

PubMed Central

Das, Amit; Chakrabarti, J.; Ghosh, Mahua

2013-01-01

We extract the thermodynamics of conformational changes in biomacromolecular complexes from the distributions of the dihedral angles of the macromolecules. These distributions are obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. The conformational thermodynamics data we obtained for calmodulin-peptide complexes using our methodology corroborate well with the experimentally observed conformational and binding entropies. The conformational free-energy changes and their contributions for different peptide-binding regions of calmodulin are evaluated microscopically. PMID:23528087

Familial aggregation analysis of gene expressions

PubMed Central

Rao, Shao-Qi; Xu, Liang-De; Zhang, Guang-Mei; Li, Xia; Li, Lin; Shen, Gong-Qing; Jiang, Yang; Yang, Yue-Ying; Gong, Bin-Sheng; Jiang, Wei; Zhang, Fan; Xiao, Yun; Wang, Qing K

2007-01-01

Traditional studies of familial aggregation are aimed at defining the genetic (and non-genetic) causes of a disease from physiological or clinical traits. However, there has been little attempt to use genome-wide gene expressions, the direct phenotypic measures of genes, as the traits to investigate several extended issues regarding the distributions of familially aggregated genes on chromosomes or in functions. In this study we conducted a genome-wide familial aggregation analysis by using the in vitro cell gene expressions of 3300 human autosome genes (Problem 1 data provided to Genetic Analysis Workshop 15) in order to answer three basic genetics questions. First, we investigated how gene expressions aggregate among different types (degrees) of relative pairs. Second, we conducted a bioinformatics analysis of highly familially aggregated genes to see how they are distributed on chromosomes. Third, we performed a gene ontology enrichment test of familially aggregated genes to find evidence to support their functional consensus. The results indicated that 1) gene expressions did aggregate in families, especially between sibs. Of 3300 human genes analyzed, there were a total of 1105 genes with one or more significant (empirical p < 0.05) familial correlation; 2) there were several genomic hot spots where highly familially aggregated genes (e.g., the chromosome 6 HLA genes cluster) were clustered; 3) as we expected, gene ontology enrichment tests revealed that the 1105 genes were aggregating not only in families but also in functional categories. PMID:18466548

Lineage-specific expansion of IFIT gene family: an insight into coevolution with IFN gene family.

PubMed

Liu, Ying; Zhang, Yi-Bing; Liu, Ting-Kai; Gui, Jian-Fang

2013-01-01

In mammals, IFIT (Interferon [IFN]-induced proteins with Tetratricopeptide Repeat [TPR] motifs) family genes are involved in many cellular and viral processes, which are tightly related to mammalian IFN response. However, little is known about non-mammalian IFIT genes. In the present study, IFIT genes are identified in the genome databases from the jawed vertebrates including the cartilaginous elephant shark but not from non-vertebrates such as lancelet, sea squirt and acorn worm, suggesting that IFIT gene family originates from a vertebrate ancestor about 450 million years ago. IFIT family genes show conserved gene structure and gene arrangements. Phylogenetic analyses reveal that this gene family has expanded through lineage-specific and species-specific gene duplication. Interestingly, IFN gene family seem to share a common ancestor and a similar evolutionary mechanism; the function link of IFIT genes to IFN response is present early since the origin of both gene families, as evidenced by the finding that zebrafish IFIT genes are upregulated by fish IFNs, poly(I:C) and two transcription factors IRF3/IRF7, likely via the IFN-stimulated response elements (ISRE) within the promoters of vertebrate IFIT family genes. These coevolution features creates functional association of both family genes to fulfill a common biological process, which is likely selected by viral infection during evolution of vertebrates. Our results are helpful for understanding of evolution of vertebrate IFN system.

Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode.

PubMed

Matthews, Benjamin F; Beard, Hunter; MacDonald, Margaret H; Kabir, Sara; Youssef, Reham M; Hosseini, Parsa; Brewer, Eric

2013-05-01

During pathogen attack, the host plant induces genes to ward off the pathogen while the pathogen often produces effector proteins to increase susceptibility of the host. Gene expression studies of syncytia formed in soybean root by soybean cyst nematode (Heterodera glycines) identified many genes altered in expression in resistant and susceptible roots. However, it is difficult to assess the role and impact of these genes on resistance using gene expression patterns alone. We selected 100 soybean genes from published microarray studies and individually overexpressed them in soybean roots to determine their impact on cyst nematode development. Nine genes reduced the number of mature females by more than 50 % when overexpressed, including genes encoding ascorbate peroxidase, β-1,4-endoglucanase, short chain dehydrogenase, lipase, DREPP membrane protein, calmodulin, and three proteins of unknown function. One gene encoding a serine hydroxymethyltransferase decreased the number of mature cyst nematode females by 45 % and is located at the Rhg4 locus. Four genes increased the number of mature cyst nematode females by more than 200 %, while thirteen others increased the number of mature cyst nematode females by more than 150 %. Our data support a role for auxin and ethylene in susceptibility of soybean to cyst nematodes. These studies highlight the contrasting gene sets induced by host and nematode during infection and provide new insights into the interactions between host and pathogen at the molecular level. Overexpression of some of these genes result in a greater decrease in the number of cysts formed than recognized soybean cyst nematode resistance loci.

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

NASA Technical Reports Server (NTRS)

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.

Molecular modeling of calmodulin: a comparison with crystallographic data

NASA Technical Reports Server (NTRS)

McDonald, J. J.; Rein, R.

1989-01-01

Two methods of side-chain placement on a modeled protein have been examined. Two molecular models of calmodulin were constructed that differ in the treatment of side chains prior to optimization of the molecule. A virtual bond analysis program developed by Purisima and Scheraga was used to determine the backbone conformation based on 2.2 angstroms resolution C alpha coordinates for the molecules. In the first model, side chains were initially constructed in an extended conformation. In the second model, a conformational grid search technique was employed. Calcium ions were treated explicitly during energy optimization using CHARMM. The models are compared to a recently published refined crystal structure of calmodulin. The results indicate that the initial choices for side-chains, but also significant effects on the main-chain conformation and supersecondary structure. The conformational differences are discussed. Analysis of these and other methods makes possible the formulation of a methodology for more appropriate side-chain placement in modeled proteins.

Inhibition of mechanotransducer currents in crayfish sensory neuron by CGS 9343B, a calmodulin antagonist.

PubMed

Lin, J H; Rydqvist, B

2000-05-26

The effects of CGS 9343B (zaldaride maleate), a calmodulin antagonist, on mechanosensitive channels were examined in crayfish slowly adapting sensory neurons using the two-electrode voltage clamp technique. In addition to its inhibition of voltage-gated Na(+) and K(+) currents, CGS 9343B (<30 microM) blocked reversibly the receptor current in a dose-dependent and voltage-dependent manner with a dissociation constant (K(d)) of 26.8 microM. The time course of the block was 265 s. Within the extension range of 3-30%, the reduction in receptor current was stimulus-independent and the gating mechanisms were not affected. Extracellular Ca(2+) was not necessary for its blocking effects. No changes in passive muscle tension were observed in the presence of 20 microM CGS 9343B. These results suggest that CGS 9343B, as a calmodulin antagonist, can also block mechanosensitive channels, possibly by being incorporated into the lipid membrane and/or interacting with the channel protein.

A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin-dependent kinase II holoenzyme

PubMed Central

Chao, Luke H.; Stratton, Margaret M.; Lee, Il-Hyung; Rosenberg, Oren S.; Levitz, Joshua; Mandell, Daniel J.; Kortemme, Tanja; Groves, Jay T.; Schulman, Howard; Kuriyan, John

2011-01-01

Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains. PMID:21884935

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

PubMed Central

Rodríguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Akerboom, Jasper; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

2008-01-01

Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way. PMID:18607093

Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin.

PubMed

Miclet, Emeric; Bourgoin-Voillard, Sandrine; Byrne, Cillian; Jacquot, Yves

2016-01-01

The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17β-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca(2+)-calmodulin, a protein also highly structured in α-helix, is a key coactivator for ERα activity. Here, we show how circular dichroism can be used to study the interaction of ERα with Ca(2+)-calmodulin. Our approach allows the determination not only of the conformational changes induced upon complex formation but also the dissociation constant (K d) of this interaction.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

PubMed

Sucharov, Carmen C; Mariner, Peter D; Nunley, Karin R; Long, Carlin; Leinwand, Leslie; Bristow, Michael R

2006-09-01

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein.

PubMed

González-Andrade, Martín; Figueroa, Mario; Rodríguez-Sotres, Rogelio; Mata, Rachel; Sosa-Peinado, Alejandro

2009-04-01

This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change ( approximately 80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC(50) values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.

Calcium/Calmodulin-dependent Protein Kinase II is a Ubiquitous Molecule in Human Long-term Memory Synaptic Plasticity: A Systematic Review

PubMed Central

Ataei, Negar; Sabzghabaee, Ali Mohammad; Movahedian, Ahmad

2015-01-01

Background: Long-term memory is based on synaptic plasticity, a series of biochemical mechanisms include changes in structure and proteins of brain's neurons. In this article, we systematically reviewed the studies that indicate calcium/calmodulin kinase II (CaMKII) is a ubiquitous molecule among different enzymes involved in human long-term memory and the main downstream signaling pathway of long-term memory. Methods: All of the observational, case–control and review studies were considered and evaluated by the search engines PubMed, Cochrane Central Register of Controlled Trials and ScienceDirect Scopus between 1990 and February 2015. We did not carry out meta-analysis. Results: At the first search, it was fined 1015 articles which included “synaptic plasticity” OR “neuronal plasticity” OR “synaptic density” AND memory AND “molecular mechanism” AND “calcium/calmodulin-dependent protein kinase II” OR CaMKII as the keywords. A total of 335 articles were duplicates in the databases and eliminated. A total of 680 title articles were evaluated. Finally, 40 articles were selected as reference. Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory. PMID:26445635

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

NASA Technical Reports Server (NTRS)

Hong, B.; Ichida, A.; Wang, Y.; Gens, J. S.; Pickard, B. G.; Harper, J. F.; Evans, M. L. (Principal Investigator)

1999-01-01

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

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

PubMed

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.

The Caenorhabditis chemoreceptor gene families.

PubMed

Thomas, James H; Robertson, Hugh M

2008-10-06

Chemoreceptor proteins mediate the first step in the transduction of environmental chemical stimuli, defining the breadth of detection and conferring stimulus specificity. Animal genomes contain families of genes encoding chemoreceptors that mediate taste, olfaction, and pheromone responses. The size and diversity of these families reflect the biology of chemoperception in specific species. Based on manual curation and sequence comparisons among putative G-protein-coupled chemoreceptor genes in the nematode Caenorhabditis elegans, we identified approximately 1300 genes and 400 pseudogenes in the 19 largest gene families, most of which fall into larger superfamilies. In the related species C. briggsae and C. remanei, we identified most or all genes in each of the 19 families. For most families, C. elegans has the largest number of genes and C. briggsae the smallest number, suggesting changes in the importance of chemoperception among the species. Protein trees reveal family-specific and species-specific patterns of gene duplication and gene loss. The frequency of strict orthologs varies among the families, from just over 50% in two families to less than 5% in three families. Several families include large species-specific expansions, mostly in C. elegans and C. remanei. Chemoreceptor gene families in Caenorhabditis species are large and evolutionarily dynamic as a result of gene duplication and gene loss. These dynamics shape the chemoreceptor gene complements in Caenorhabditis species and define the receptor space available for chemosensory responses. To explain these patterns, we propose the gray pawn hypothesis: individual genes are of little significance, but the aggregate of a large number of diverse genes is required to cover a large phenotype space.

The Caenorhabditis chemoreceptor gene families

PubMed Central

Thomas, James H; Robertson, Hugh M

2008-01-01

Background Chemoreceptor proteins mediate the first step in the transduction of environmental chemical stimuli, defining the breadth of detection and conferring stimulus specificity. Animal genomes contain families of genes encoding chemoreceptors that mediate taste, olfaction, and pheromone responses. The size and diversity of these families reflect the biology of chemoperception in specific species. Results Based on manual curation and sequence comparisons among putative G-protein-coupled chemoreceptor genes in the nematode Caenorhabditis elegans, we identified approximately 1300 genes and 400 pseudogenes in the 19 largest gene families, most of which fall into larger superfamilies. In the related species C. briggsae and C. remanei, we identified most or all genes in each of the 19 families. For most families, C. elegans has the largest number of genes and C. briggsae the smallest number, suggesting changes in the importance of chemoperception among the species. Protein trees reveal family-specific and species-specific patterns of gene duplication and gene loss. The frequency of strict orthologs varies among the families, from just over 50% in two families to less than 5% in three families. Several families include large species-specific expansions, mostly in C. elegans and C. remanei. Conclusion Chemoreceptor gene families in Caenorhabditis species are large and evolutionarily dynamic as a result of gene duplication and gene loss. These dynamics shape the chemoreceptor gene complements in Caenorhabditis species and define the receptor space available for chemosensory responses. To explain these patterns, we propose the gray pawn hypothesis: individual genes are of little significance, but the aggregate of a large number of diverse genes is required to cover a large phenotype space. PMID:18837995

Global differential expression of genes located in the Down Syndrome Critical Region in normal human brain

PubMed Central

Montoya, Julio Cesar; Fajardo, Dianora; Peña, Angela; Sánchez, Adalberto; Domínguez, Martha C; Satizábal, José María

2014-01-01

Background: The information of gene expression obtained from databases, have made possible the extraction and analysis of data related with several molecular processes involving not only in brain homeostasis but its disruption in some neuropathologies; principally in Down syndrome and the Alzheimer disease. Objective: To correlate the levels of transcription of 19 genes located in the Down Syndrome Critical Region (DSCR) with their expression in several substructures of normal human brain. Methods: There were obtained expression profiles of 19 DSCR genes in 42 brain substructures, from gene expression values available at the database of the human brain of the Brain Atlas of the Allen Institute for Brain Sciences", (http://human.brain-map.org/). The co-expression patterns of DSCR genes in brain were calculated by using multivariate statistical methods. Results: Highest levels of gene expression were registered at caudate nucleus, nucleus accumbens and putamen among central areas of cerebral cortex. Increased expression levels of RCAN1 that encode by a protein involved in signal transduction process of the CNS were recorded for PCP4 that participates in the binding to calmodulin and TTC3; a protein that is associated with differentiation of neurons. That previously identified brain structures play a crucial role in the learning process, in different class of memory and in motor skills. Conclusion: The precise regulation of DSCR gene expression is crucial to maintain the brain homeostasis, especially in those areas with high levels of gene expression associated with a remarkable process of learning and cognition. PMID:25767303

Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA.

PubMed

Tamori, Akihiro; Yamanishi, Yoshihiro; Kawashima, Shuichi; Kanehisa, Minoru; Enomoto, Masaru; Tanaka, Hiromu; Kubo, Shoji; Shiomi, Susumu; Nishiguchi, Shuhei

2005-08-15

Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA. We examined 15 cases of HCC infected with HBV by cassette ligation-mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis. The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes. HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site-specific expression of such genes during hepatocarcinogenesis.

Defining global neuroendocrine gene expression patterns associated with reproductive seasonality in fish.

PubMed

Zhang, Dapeng; Xiong, Huiling; Mennigen, Jan A; Popesku, Jason T; Marlatt, Vicki L; Martyniuk, Christopher J; Crump, Kate; Cossins, Andrew R; Xia, Xuhua; Trudeau, Vance L

2009-06-05

Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABA(A) gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.

Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish

PubMed Central

Mennigen, Jan A.; Popesku, Jason T.; Marlatt, Vicki L.; Martyniuk, Christopher J.; Crump, Kate; Cossins, Andrew R.; Xia, Xuhua; Trudeau, Vance L.

2009-01-01

Background Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. Methodology/Principal Findings In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABAA gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. Conclusions/Significance Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development. PMID:19503831

Altered Gene Expression in Three Plant Species in Response to Treatment with Nep1, a Fungal Protein That Causes Necrosis

PubMed Central

Keates, Sarah E.; Kostman, Todd A.; Anderson, James D.; Bailey, Bryan A.

2003-01-01

Nep1 is an extracellular fungal protein that causes necrosis when applied to many dicotyledonous plants, including invasive weed species. Using transmission electron microscopy, it was determined that application of Nep1 (1.0 μg mL–1, 0.1% [v/v] Silwet-L77) to Arabidopsis and two invasive weed species, spotted knapweed (Centaurea maculosa) and dandelion (Taraxacum officinale), caused a reduction in the thickness of the cuticle and a breakdown of chloroplasts 1 to 4 h after treatment. Membrane breakdown was most severe in cells closest to the surface of application. Differential display was used to isolate cDNA clones from the three species showing differential expression in response to Nep1 treatment. Differential gene expression was observed for a putative serpin (CmSER-1) and a calmodulin-like (CmCAL-1) protein from spotted knapweed, and a putative protein phosphatase 2C (ToPP2C-1) and cytochrome P-450 (ToCYP-1) protein from dandelion. In addition, differential expression was observed for genes coding for a putative protein kinase (AtPK-1), a homolog (AtWI-12) of wound-induced WI12, a homolog (AtLEA-1) of late embryogenesis abundant LEA-5, a WRKY-18 DNA-binding protein (AtWRKY-18), and a phospholipase D (AtPLD-1) from Arabidopsis. Genes showing elevated mRNA levels in Nep1-treated (5 μg mL–1, 0.1% [v/v] Silwet-L77) leaves 15 min after Nep1 treatment included CmSER-1 and CmCAL-1 for spotted knapweed, ToCYP-1 and CmCAL-1 for dandelion, and AtPK-1, AtWRKY-18, AtWI-12, and AtLEA-1 for Arabidopsis. Levels of mRNA for AtPLD-1 (Arabidopsis) and ToPP2C-1 (dandelion) decreased rapidly in Silwet-l77-treated plants between 15 min and 4 h of treatment, but were maintained or decreased more slowly over time in Nep1-treated (5 μg mL–1, 0.1% [v/v] Silwet-L77) leaves. In general, increases in mRNA band intensities were in the range of two to five times, with only ToCYP-1 in dandelion exceeding an increase of 10 times. The identified genes have been shown to be involved or are related to gene families that are involved in plant stress responses, including wounding, drought, senescence, and disease resistance. PMID:12857840

Inferring Gene Family Histories in Yeast Identifies Lineage Specific Expansions

PubMed Central

Ames, Ryan M.; Money, Daniel; Lovell, Simon C.

2014-01-01

The complement of genes found in the genome is a balance between gene gain and gene loss. Knowledge of the specific genes that are gained and lost over evolutionary time allows an understanding of the evolution of biological functions. Here we use new evolutionary models to infer gene family histories across complete yeast genomes; these models allow us to estimate the relative genome-wide rates of gene birth, death, innovation and extinction (loss of an entire family) for the first time. We show that the rates of gene family evolution vary both between gene families and between species. We are also able to identify those families that have experienced rapid lineage specific expansion/contraction and show that these families are enriched for specific functions. Moreover, we find that families with specific functions are repeatedly expanded in multiple species, suggesting the presence of common adaptations and that these family expansions/contractions are not random. Additionally, we identify potential specialisations, unique to specific species, in the functions of lineage specific expanded families. These results suggest that an important mechanism in the evolution of genome content is the presence of lineage-specific gene family changes. PMID:24921666

Algal ancestor of land plants was preadapted for symbiosis.

PubMed

Delaux, Pierre-Marc; Radhakrishnan, Guru V; Jayaraman, Dhileepkumar; Cheema, Jitender; Malbreil, Mathilde; Volkening, Jeremy D; Sekimoto, Hiroyuki; Nishiyama, Tomoaki; Melkonian, Michael; Pokorny, Lisa; Rothfels, Carl J; Sederoff, Heike Winter; Stevenson, Dennis W; Surek, Barbara; Zhang, Yong; Sussman, Michael R; Dunand, Christophe; Morris, Richard J; Roux, Christophe; Wong, Gane Ka-Shu; Oldroyd, Giles E D; Ané, Jean-Michel

2015-10-27

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

Comparison of Long Noncoding RNA and mRNA Expression Profiles in Mesenchymal Stem Cells Derived from Human Periodontal Ligament and Bone Marrow

PubMed Central

Dong, Rui; Du, Juan; Wang, Liping; Wang, Jinsong; Ding, Gang; Wang, Songlin; Fan, Zhipeng

2014-01-01

Mesenchymal stem cells (MSCs) in different anatomic locations possess diverse biological activities. Maintaining the pluripotent state and differentiation depend on the expression and regulation of thousands of genes, but it remains unclear which molecular mechanisms underlie MSC diversity. Thus, potential MSC applications are restricted. Long noncoding RNAs (lncRNAs) are implicated in the complex molecular circuitry of cellular processes. We investigated differences in lncRNA and mRNA expression profiles between bone marrow stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs) with lncRNA microarray assays and bioinformatics analysis. In PDLSCs, numerous lncRNAs were significantly upregulated (n = 457) or downregulated (n = 513) compared to BMSCs. Furthermore, 1,578 mRNAs were differentially expressed. These genes implicated cellular pathways that may be associated with MSC characteristics, including apoptosis, MAPK, cell cycle, and Wnt signaling pathway. Signal-net analysis indicated that phospholipase C beta 4, filamin B beta, calcium/calmodulin-dependent protein kinase II gamma, and the ionotropic glutamate receptor, AMPA 1, had the highest betweenness centrality among significant genes in the differential gene profile network. A comparison between the coding-noncoding gene coexpression networks of PDLSCs and BMSCs identified chemokine (C-X-C motif) ligand 12 as a core regulatory factor in MSC biology. These results provided insight into the mechanisms underlying MSC biology. PMID:24790996

Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Hidaka, H.

1993-01-01

Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

Constitutive Expression of Mammalian Nitric Oxide Synthase in Tobacco Plants Triggers Disease Resistance to Pathogens

PubMed Central

Chun, Hyun Jin; Park, Hyeong Cheol; Koo, Sung Cheol; Lee, Ju Huck; Park, Chan Young; Choi, Man Soo; Kang, Chang Ho; Baek, Dongwon; Cheong, Yong Hwa; Yun, Dae-Jin; Chung, Woo Sik; Cho, Moo Je; Kim, Min Chul

2012-01-01

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H2O2, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance. PMID:23124383

Evolutionary dynamics of olfactory and other chemosensory receptor genes in vertebrates

PubMed Central

Niimura, Yoshihito

2007-01-01

The numbers of functional olfactory receptor (OR) genes in humans and mice are about 400 and 1,000 respectively. In both humans and mice, these genes exist as genomic clusters and are scattered over almost all chromosomes. The difference in the number of genes between the two species is apparently caused by massive inactivation of OR genes in the human lineage and a substantial increase of OR genes in the mouse lineage after the human–mouse divergence. Compared with mammals, fishes have a much smaller number of OR genes. However, the OR gene family in fishes is much more divergent than that in mammals. Fishes have many different groups of genes that are absent in mammals, suggesting that the mammalian OR gene family is characterized by the loss of many group genes that existed in the ancestor of vertebrates and the subsequent expansion of specific groups of genes. Therefore, this gene family apparently changed dynamically depending on the evolutionary lineage and evolved under the birth-and-death model of evolution. Study of the evolutionary changes of two gene families for vomeronasal receptors and two gene families for taste receptors, which are structurally similar, but remotely related to OR genes, showed that some of the gene families evolved in the same fashion as the OR gene family. It appears that the number and types of genes in chemosensory receptor gene families have evolved in response to environmental needs, but they are also affected by fortuitous factors. PMID:16607462

Comparative genome analysis of PHB gene family reveals deep evolutionary origins and diverse gene function.

PubMed

Di, Chao; Xu, Wenying; Su, Zhen; Yuan, Joshua S

2010-10-07

PHB (Prohibitin) gene family is involved in a variety of functions important for different biological processes. PHB genes are ubiquitously present in divergent species from prokaryotes to eukaryotes. Human PHB genes have been found to be associated with various diseases. Recent studies by our group and others have shown diverse function of PHB genes in plants for development, senescence, defence, and others. Despite the importance of the PHB gene family, no comprehensive gene family analysis has been carried to evaluate the relatedness of PHB genes across different species. In order to better guide the gene function analysis and understand the evolution of the PHB gene family, we therefore carried out the comparative genome analysis of the PHB genes across different kingdoms. The relatedness, motif distribution, and intron/exon distribution all indicated that PHB genes is a relatively conserved gene family. The PHB genes can be classified into 5 classes and each class have a very deep evolutionary origin. The PHB genes within the class maintained the same motif patterns during the evolution. With Arabidopsis as the model species, we found that PHB gene intron/exon structure and domains are also conserved during the evolution. Despite being a conserved gene family, various gene duplication events led to the expansion of the PHB genes. Both segmental and tandem gene duplication were involved in Arabidopsis PHB gene family expansion. However, segmental duplication is predominant in Arabidopsis. Moreover, most of the duplicated genes experienced neofunctionalization. The results highlighted that PHB genes might be involved in important functions so that the duplicated genes are under the evolutionary pressure to derive new function. PHB gene family is a conserved gene family and accounts for diverse but important biological functions based on the similar molecular mechanisms. The highly diverse biological function indicated that more research needs to be carried out to dissect the PHB gene function. The conserved gene evolution indicated that the study in the model species can be translated to human and mammalian studies.

Evolution, functional differentiation, and co-expression of the RLK gene family revealed in Jilin ginseng, Panax ginseng C.A. Meyer.

PubMed

Lin, Yanping; Wang, Kangyu; Li, Xiangyu; Sun, Chunyu; Yin, Rui; Wang, Yanfang; Wang, Yi; Zhang, Meiping

2018-02-21

Most genes in a genome exist in the form of a gene family; therefore, it is necessary to have knowledge of how a gene family functions to comprehensively understand organismal biology. The receptor-like kinase (RLK)-encoding gene family is one of the most important gene families in plants. It plays important roles in biotic and abiotic stress tolerances, and growth and development. However, little is known about the functional differentiation and relationships among the gene members within a gene family in plants. This study has isolated 563 RLK genes (designated as PgRLK genes) expressed in Jilin ginseng (Panax ginseng C.A. Meyer), investigated their evolution, and deciphered their functional diversification and relationships. The PgRLK gene family is highly diverged and formed into eight types. The LRR type is the earliest and most prevalent, while only the Lec type originated after P. ginseng evolved. Furthermore, although the members of the PgRLK gene family all encode receptor-like protein kinases and share conservative domains, they are functionally very diverse, participating in numerous biological processes. The expressions of different members of the PgRLK gene family are extremely variable within a tissue, at a developmental stage and in the same cultivar, but most of the genes tend to express correlatively, forming a co-expression network. These results not only provide a deeper and comprehensive understanding of the evolution, functional differentiation and correlation of a gene family in plants, but also an RLK genic resource useful for enhanced ginseng genetic improvement.

Ca(2+)-related signaling and protein phosphorylation abnormalities play central roles in a new experimental model of electrical storm.

PubMed

Tsuji, Yukiomi; Hojo, Mayumi; Voigt, Niels; El-Armouche, Ali; Inden, Yasuya; Murohara, Toyoaki; Dobrev, Dobromir; Nattel, Stanley; Kodama, Itsuo; Kamiya, Kaichiro

2011-05-24

Electrical storm (ES), characterized by recurrent ventricular tachycardia/fibrillation, typically occurs in implantable cardioverter-defibrillator patients and adversely affects prognosis. However, the underlying molecular basis is poorly understood. In the present study, we report a new experimental model featuring repetitive episodes of implantable cardioverter-defibrillator firing for recurrent ventricular fibrillation (VF), in which we assessed involvement of Ca(2+)-related protein alterations in ES. We studied 37 rabbits with complete atrioventricular block for ≈80 days, all with implantable cardioverter-defibrillator implantation. All rabbits showed long-QT and VF episodes. Fifty-three percent of rabbits developed ES (≥3 VF episodes per 24-hour period; 103±23 VF episodes per rabbit). Expression/phosphorylation of Ca(2+)-handling proteins was assessed in left ventricular tissues from rabbits with the following: ES; VF episodes but not ES (non-ES); and controls. Left ventricular end-diastolic diameter increased comparably in ES and non-ES rabbits, but contractile dysfunction was significantly greater in ES than in non-ES rabbits. ES rabbits showed striking hyperphosphorylation of Ca(2+)/calmodulin-dependent protein kinase II, prominent phospholamban dephosphorylation, and increased protein phosphatase 1 and 2A expression versus control and non-ES rabbits. Ryanodine receptors were similarly hyperphosphorylated at Ser2815 in ES and non-ES rabbits, but ryanodine receptor Ser2809 and L-type Ca(2+) channel α-subunit hyperphosphorylation were significantly greater in ES versus non-ES rabbits. To examine direct effects of repeated VF/defibrillation, VF was induced 10 times in control rabbits. Repeated VF tissues showed autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II upregulation and phospholamban dephosphorylation like those of ES rabbit hearts. Continuous infusion of a calmodulin antagonist (W-7) to ES rabbits reduced Ca(2+)/calmodulin-dependent protein kinase II hyperphosphorylation, suppressed ventricular tachycardia/fibrillation, and rescued left ventricular dysfunction. ES causes Ca(2+)/calmodulin-dependent protein kinase II activation and phospholamban dephosphorylation, which can explain the vicious cycle of arrhythmia promotion and mechanical dysfunction that characterizes ES.

Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

PubMed Central

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Background Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the “cytoplasmic” myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. Methodology/Principal Findings We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. Conclusions/Significance We have shown that the novel specific NLS brings to the cell nucleus not only the “nuclear” isoform of myosin I (NM1 protein) but also its “cytoplasmic” isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus. PMID:22295092

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

NASA Technical Reports Server (NTRS)

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.

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

PubMed

Wei, Yu-Ping; Ye, Jin-Wang; Wang, Xiong; Zhu, Li-Ping; Hu, Qing-Hua; Wang, Qun; Ke, Dan; Tian, Qing; Wang, Jian-Zhi

2018-04-01

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca 2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca 2+ concentration with a simultaneous increase in the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca 2+ /CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca 2+ /calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca 2+ /CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca 2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

Treatment of skin injuries induced by sulfur mustard with calmodulin antagonists, using the pig model.

PubMed

Kadar, T; Fishbeine, E; Meshulam, Y; Sahar, R; Chapman, S; Liani, H; Barness, I; Amir, A

2000-12-01

Sulfur mustard (HD) is a potent cutaneous vesicant that penetrates rapidly through the skin, causing prolonged injuries and leading to severe incapacitation. Although there has been long and intensive efforts to find a treatment for HD skin lesions, no effective treatment is available for HD-induced skin injuries. Recently, ointments containing calmodulin antagonists were found to be effective in preventing skin injuries induced by HD in hairless mice. The present study was designed to investigate the beneficial effects of topical treatments with calmodulin antagonists against HD skin lesions in the pig model. The pig is used as a preferred animal model for human skin in many studies, including vesicants. Neat HD, either in liquid form (0.2-1 microl droplets) or as vapour, was applied to the back skin of female pigs (a cross Large White & Landrace, 10-12 kg) for various exposure durations. Evaluation was based on quantitative analysis of the degree of erythema and area of the lesions, as well as histological evaluation. Calmodulin antagonists (10% pentamide, 1% trifluoperazine, 2% thioridazine) and anaesthetics (20% lidocaine and 3% benoxinate) were dissolved in pluronic F-127 base according to Kim et al. (Eur. J. Pharmacol. 1996; 313: 107-114) or in saline, and were applied either topically as ointments or by intradermal injection, as early as 5 min post-exposure (twice a day for at least 3 days). The results demonstrated that topically applied pluronic base ointments containing lidocaine or pentamide produce beneficial effects when applied immediately after short-term HD exposure to pig skin.

Genome structure drives patterns of gene family evolution in ciliates, a case study using Chilodonella uncinata (Protista, Ciliophora, Phyllopharyngea)

PubMed Central

Gao, Feng; Song, Weibo; Katz, Laura A.

2014-01-01

In most lineages, diversity among gene family members results from gene duplication followed by sequence divergence. Because of the genome rearrangements during the development of somatic nuclei, gene family evolution in ciliates involves more complex processes. Previous work on the ciliate Chilodonella uncinata revealed that macronuclear β-tubulin gene family members are generated by alternative processing, in which germline regions are alternatively used in multiple macronuclear chromosomes. To further study genome evolution in this ciliate, we analyzed its transcriptome and found that: 1) alternative processing is extensive among gene families; and 2) such gene families are likely to be C. uncinata-specific. We characterized additional macronuclear and micronuclear copies of one candidate alternatively processed gene family -- a protein kinase domain containing protein (PKc) -- from two C. uncinata strains. Analysis of the PKc sequences reveals: 1) multiple PKc gene family members in the macronucleus share some identical regions flanked by divergent regions; and 2) the shared identical regions are processed from a single micronuclear chromosome. We discuss analogous processes in lineages across the eukaryotic tree of life to provide further insights on the impact of genome structure on gene family evolution in eukaryotes. PMID:24749903

Numbers of genes in the NBS and RLK families vary by more than four-fold within a plant species and are regulated by multiple factors.

PubMed

Zhang, Meiping; Wu, Yen-Hsuan; Lee, Mi-Kyung; Liu, Yun-Hua; Rong, Ying; Santos, Teofila S; Wu, Chengcang; Xie, Fangming; Nelson, Randall L; Zhang, Hong-Bin

2010-10-01

Many genes exist in the form of families; however, little is known about their size variation, evolution and biology. Here, we present the size variation and evolution of the nucleotide-binding site (NBS)-encoding gene family and receptor-like kinase (RLK) gene family in Oryza, Glycine and Gossypium. The sizes of both families vary by numeral fold, not only among species, surprisingly, also within a species. The size variations of the gene families are shown to correlate with each other, indicating their interactions, and driven by natural selection, artificial selection and genome size variation, but likely not by polyploidization. The numbers of genes in the families in a polyploid species are similar to those of one of its diploid donors, suggesting that polyploidization plays little roles in the expansion of the gene families and that organisms tend not to maintain their 'surplus' genes in the course of evolution. Furthermore, it is found that the size variations of both gene families are associated with organisms' phylogeny, suggesting their roles in speciation and evolution. Since both selection and speciation act on organism's morphological, physiological and biological variation, our results indicate that the variation of gene family size provides a source of genetic variation and evolution.

GeneSeqToFamily: a Galaxy workflow to find gene families based on the Ensembl Compara GeneTrees pipeline.

PubMed

Thanki, Anil S; Soranzo, Nicola; Haerty, Wilfried; Davey, Robert P

2018-03-01

Gene duplication is a major factor contributing to evolutionary novelty, and the contraction or expansion of gene families has often been associated with morphological, physiological, and environmental adaptations. The study of homologous genes helps us to understand the evolution of gene families. It plays a vital role in finding ancestral gene duplication events as well as identifying genes that have diverged from a common ancestor under positive selection. There are various tools available, such as MSOAR, OrthoMCL, and HomoloGene, to identify gene families and visualize syntenic information between species, providing an overview of syntenic regions evolution at the family level. Unfortunately, none of them provide information about structural changes within genes, such as the conservation of ancestral exon boundaries among multiple genomes. The Ensembl GeneTrees computational pipeline generates gene trees based on coding sequences, provides details about exon conservation, and is used in the Ensembl Compara project to discover gene families. A certain amount of expertise is required to configure and run the Ensembl Compara GeneTrees pipeline via command line. Therefore, we converted this pipeline into a Galaxy workflow, called GeneSeqToFamily, and provided additional functionality. This workflow uses existing tools from the Galaxy ToolShed, as well as providing additional wrappers and tools that are required to run the workflow. GeneSeqToFamily represents the Ensembl GeneTrees pipeline as a set of interconnected Galaxy tools, so they can be run interactively within the Galaxy's user-friendly workflow environment while still providing the flexibility to tailor the analysis by changing configurations and tools if necessary. Additional tools allow users to subsequently visualize the gene families produced by the workflow, using the Aequatus.js interactive tool, which has been developed as part of the Aequatus software project.

Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize [Phosphorylation of a NAC Transcription Factor by ZmCCaMK Regulates Abscisic Acid-Induced Antioxidant Defense in Maize

DOE PAGES

Zhu, Yuan; Yan, Jingwei; Liu, Weijuan; ...

2016-05-10

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize, which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 display a partially overlapping expression pattern with ZmCCaMK after ABA treatment and H 2O 2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates S113 of ZmNAC84 inmore » vitro, and S113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco can improve drought tolerance, and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H 2O 2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK, and subsequently the activated ZmCCaMK phosphorylates ZmNAC84 at S113, thereby inducing antioxidant defense by activating downstream genes.« less

Regulation of Polycystin-1 Function by Calmodulin Binding

PubMed Central

Doerr, Nicholas; Wang, Yidi; Kipp, Kevin R.; Liu, Guangyi; Benza, Jesse J.; Pletnev, Vladimir; Pavlov, Tengis S.; Staruschenko, Alexander; Mohieldin, Ashraf M.; Takahashi, Maki; Nauli, Surya M.; Weimbs, Thomas

2016-01-01

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM. PMID:27560828

Regulation of a phenylalanine ammonia lyase (BbPAL) by calmodulin in response to environmental changes in the entomopathogenic fungus Beauveria bassiana.

PubMed

Kim, Jiyoung; Park, Hyesung; Han, Jae-Gu; Oh, Junsang; Choi, Hyung-Kyoon; Kim, Seong Hwan; Sung, Gi-Ho

2015-11-01

Phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5) catalyses the deamination of L -phenylalanine to trans-cinnamic acid and ammonia, facilitating a critical step in the phenylpropanoid pathway that produces a variety of secondary metabolites. In this study, we isolated BbPAL gene in the entomopathogenic fungus Beauveria bassiana. According to multiple sequence alignment, homology modelling and in vitro PAL activity, we demonstrated that BbPAL acts as a typical PAL enzyme in B. bassiana. BbPAL interacted with calmodulin (CaM) in vitro and in vivo, indicating that BbPAL is a novel CaM-binding protein. The functional role of CaM in BbPAL action was to negatively regulate the BbPAL activity in B. bassiana. High-performance liquid chromatography analysis revealed that L -phenylalanine was reduced and trans-cinnamic acid was increased in response to the CaM inhibitor W-7. Dark conditions suppressed BbPAL activity in B. bassiana, compared with light. In addition, heat and cold stresses inhibited BbPAL activity in B. bassiana. Interestingly, these negative effects of BbPAL activity by dark, heat and cold conditions were recovered by W-7 treatment, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbPAL plays a role in the phenylpropanoid pathway mediated by environmental stimuli via the CaM signalling pathway. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

NASA Technical Reports Server (NTRS)

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.

Use of a rep-PCR system to predict species in the Aspergillus section Nigri.

PubMed

Palencia, Edwin R; Klich, Maren A; Glenn, Anthony E; Bacon, Charles W

2009-10-01

The Aspergillus niger aggregate within the A. section Nigri is a group of black-spored aspergilli of great agro-economic importance whose well defined taxonomy has been elusive. Rep-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automated rep-PCR barcoding system to distinguish morphotypic species and compare the results with the data obtained from ITS and partial calmodulin regions. For this purpose, 20 morphotyped black-spored Aspergillus species were used to create the A. section Nigri library in this barcoding system that served to identify 34 field isolates. A pair-wise similarity matrix was calculated using the cone-based Pearson correlation method and the dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA), illustrating four different clustered groups: the uniseriate cluster (I), the Aspergillus carbonarius cluster (II), and. the two A. niger aggregate clusters (named III.A and III.B). Rep-PCR showed higher resolution than the ITS and the partial calmodulin gene analytical procedures. The data of the 34 unknown field isolates, collected from different locations in the United States, indicated that only 12% of the field isolates were >95% similar to one of the genotypes included in the A. section Nigri library. However, 64% of the field isolates matched genotypes with the reference library (similarity values >90%). Based on these results, this barcoding procedure has the potential for use as a reproducible tool for identifying the black-spored aspergilli.

Gallic acid attenuates calcium calmodulin-dependent kinase II-induced apoptosis in spontaneously hypertensive rats.

PubMed

Jin, Li; Piao, Zhe Hao; Liu, Chun Ping; Sun, Simei; Liu, Bin; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kee, Hae Jin; Jeong, Myung Ho

2018-03-01

Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition-induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti-cancer, anti-calcification and anti-oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase-3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ-induced apoptosis. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize [Phosphorylation of a NAC Transcription Factor by ZmCCaMK Regulates Abscisic Acid-Induced Antioxidant Defense in Maize

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Yuan; Yan, Jingwei; Liu, Weijuan

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize, which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 display a partially overlapping expression pattern with ZmCCaMK after ABA treatment and H 2O 2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates S113 of ZmNAC84 inmore » vitro, and S113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco can improve drought tolerance, and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H 2O 2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK, and subsequently the activated ZmCCaMK phosphorylates ZmNAC84 at S113, thereby inducing antioxidant defense by activating downstream genes.« less

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

NASA Technical Reports Server (NTRS)

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.

Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L

NASA Technical Reports Server (NTRS)

Ling, V.; Assmann, S. M.

1992-01-01

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.

Bordetella Adenylate Cyclase-Hemolysin Toxins

PubMed Central

Guiso, Nicole

2017-01-01

Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented. PMID:28892012

Penicillium daejeonium sp. nov., a new species isolated from a grape and schisandra fruit in Korea.

PubMed

Sang, Hyunkyu; An, Tae-Jin; Kim, Chang Sun; Choi, Young Phil; Deng, Jian-Xin; Paul, Narayan Chandra; Sung, Gi-Ho; Yu, Seung Hun

2013-08-01

Two isolates of monoverticillate Penicillium species were collected from a grape and schisandra fruit in Korea. Multigene phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and genes encoding β-tubulin (benA) and calmodulin (cmd), as well as morphological analyses revealed that the two isolates are members of the P. sclerotiorum complex in Penicillium subgenus Aspergilloides, but different from species of the P. sclerotiorum complex. The isolates are closely related to P. cainii, P. jacksonii, and P. viticola in terms of their multigene phylogeny, but their colony and conidiophore morphologies differ from those of closely related species. The name P. daejeonium is proposed for this unclassified new species belonging to the P. sclerotiorum complex in subgenus Aspergilloides.

Aspergillus bertholletius sp. nov. from Brazil Nuts

PubMed Central

Taniwaki, Marta H.; Pitt, John I.; Iamanaka, Beatriz T.; Sartori, Daniele; Copetti, Marina V.; Balajee, Arun; Fungaro, Maria Helena P.; Frisvad, Jens C.

2012-01-01

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228). PMID:22952594

Diversification of caldesmon-linked actin cytoskeleton in cell motility

PubMed Central

Mayanagi, Taira

2011-01-01

The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions. PMID:21350330

Babesia bovis expresses Bbo-6cys-E, a member of a novel gene family that is homologous to the 6-cys family of Plasmodium

USDA-ARS?s Scientific Manuscript database

A novel Babesia bovis gene family encoding proteins with similarities to the Plasmodium 6cys protein family was identified by TBLASTN searches of the Babesia bovis genome using the sequence of the P. falciparum PFS230 protein as query, and was termed Bbo-6cys gene family. The Bbo-cys6 gene family co...

GreenPhylDB v2.0: comparative and functional genomics in plants.

PubMed

Rouard, Mathieu; Guignon, Valentin; Aluome, Christelle; Laporte, Marie-Angélique; Droc, Gaëtan; Walde, Christian; Zmasek, Christian M; Périn, Christophe; Conte, Matthieu G

2011-01-01

GreenPhylDB is a database designed for comparative and functional genomics based on complete genomes. Version 2 now contains sixteen full genomes of members of the plantae kingdom, ranging from algae to angiosperms, automatically clustered into gene families. Gene families are manually annotated and then analyzed phylogenetically in order to elucidate orthologous and paralogous relationships. The database offers various lists of gene families including plant, phylum and species specific gene families. For each gene cluster or gene family, easy access to gene composition, protein domains, publications, external links and orthologous gene predictions is provided. Web interfaces have been further developed to improve the navigation through information related to gene families. New analysis tools are also available, such as a gene family ontology browser that facilitates exploration. GreenPhylDB is a component of the South Green Bioinformatics Platform (http://southgreen.cirad.fr/) and is accessible at http://greenphyl.cirad.fr. It enables comparative genomics in a broad taxonomy context to enhance the understanding of evolutionary processes and thus tends to speed up gene discovery.

Evidence for a Regulatory Role of Calcium in Gravitropism

NASA Technical Reports Server (NTRS)

Roux, S. J.

1983-01-01

Experiments conducted to determine the cellular basis of gravitropism, the phenomenon of calcium migration following gravitropic stimulation, and the preferential accumulation of calcium in cells are described. Results of autoradiographic studies of cross sections of oat, and the pryoantimony precipitation of calcium in situ are discussed. It was found that the movement of calcium during gravimetric stimulation is a redistribution of calcium from the vacuolar regions into the cells walls. This movement requires precipitation of a calcium ATPase. The control of calcium ATPase by calmodulin and whether chlorpromazine is binding to calmodulin in plants are considered.

Genome structure drives patterns of gene family evolution in ciliates, a case study using Chilodonella uncinata (Protista, Ciliophora, Phyllopharyngea).

PubMed

Gao, Feng; Song, Weibo; Katz, Laura A

2014-08-01

In most lineages, diversity among gene family members results from gene duplication followed by sequence divergence. Because of the genome rearrangements during the development of somatic nuclei, gene family evolution in ciliates involves more complex processes. Previous work on the ciliate Chilodonella uncinata revealed that macronuclear β-tubulin gene family members are generated by alternative processing, in which germline regions are alternatively used in multiple macronuclear chromosomes. To further study genome evolution in this ciliate, we analyzed its transcriptome and found that (1) alternative processing is extensive among gene families; and (2) such gene families are likely to be C. uncinata specific. We characterized additional macronuclear and micronuclear copies of one candidate alternatively processed gene family-a protein kinase domain containing protein (PKc)-from two C. uncinata strains. Analysis of the PKc sequences reveals that (1) multiple PKc gene family members in the macronucleus share some identical regions flanked by divergent regions; and (2) the shared identical regions are processed from a single micronuclear chromosome. We discuss analogous processes in lineages across the eukaryotic tree of life to provide further insights on the impact of genome structure on gene family evolution in eukaryotes. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

Mouse Vk gene classification by nucleic acid sequence similarity.

PubMed

Strohal, R; Helmberg, A; Kroemer, G; Kofler, R

1989-01-01

Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates of V gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i.e., sequence similarity) and their organization in gene families. While mouse Igh heavy chain V region (VH) gene families are relatively well-established, a corresponding systematic classification of Igk light chain V region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of the Vk germline gene repertoire and Vk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouse Vk genes in gene families composed of members with greater than 80% overall nucleic acid sequence similarity. This classification differed in several aspects from that of VH genes: only some Vk gene families were as clearly separated (by greater than 25% sequence dissimilarity) as typical VH gene families; most Vk gene families were closely related and, in several instances, members from different families were very similar (greater than 80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications for Vk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization between VH and Vk genes.

A new approach to species delimitation in Septoria

PubMed Central

Verkley, G.J.M.; Quaedvlieg, W.; Shin, H.-D.; Crous, P.W.

2013-01-01

Septoria is a large genus of asexual morphs of Ascomycota causing leaf spot diseases of many cultivated and wild plants. Host specificity has long been a decisive criterium in species delimitation in Septoria, mainly because of the paucity of useful morphological characters and the high level of variation therein. This study aimed at improving the species delimitation of Septoria by adopting a polyphasic approach, including multilocus DNA sequencing and morphological analyses on the natural substrate and in culture. To this end 365 cultures preserved in CBS, Utrecht, The Netherlands, among which many new isolates obtained from fresh field specimens were sequenced. Herbarium material including many types was also studied. Full descriptions of the morphology in planta and in vitro are provided for 57 species. DNA sequences were generated for seven loci, viz. nuclear ITS and (partial) LSU ribosomal RNA genes, RPB2, actin, calmodulin, Btub, and EF. The robust phylogeny inferred showed that the septoria-like fungi are distributed over three main clades, establishing the genera Septoria s. str., Sphaerulina, and Caryophylloseptoria gen. nov. Nine new combinations and one species, Sphaerulina tirolensis sp. nov. were proposed. It is demonstrated that some species have wider host ranges than expected, including hosts from more than one family. Septoria protearum, previously only associated with Proteaceae was found to be also associated with host plants from six additional families of phanerogams and cryptogams. To our knowledge this is the first study to provide DNA-based evidence that multiple family-associations occur for a single species in Septoria. The distribution of host families over the phylogenetic tree showed a highly dispersed pattern for 10 host plant families, providing new insight into the evolution of these fungi. It is concluded that trans-family host jumping is a major force driving the evolution of Septoria and Sphaerulina. Taxonomic novelties: New genus - Caryophylloseptoria Verkley, Quaedvlieg & Crous; New species - Sphaerulina tirolensis Verkley, Quaedvlieg & Crous; New combinations - Caryophylloseptoria lychnidis (Desm.) Verkley, Quaedvlieg & Crous, Caryophylloseptoria silenes (Westend.) Verkley, Quaedvlieg & Crous, Caryophylloseptoria spergulae (Westend.) Verkley, Quaedvlieg & Crous, Sphaerulina aceris (Lib.) Verkley, Quaedvlieg & Crous, Sphaerulina cornicola (DC.: Fr.) Verkley, Quaedvlieg & Crous, Sphaerulina gei (Roberge ex Desm.) Verkley, Quaedvlieg & Crous, Sphaerulina hyperici (Roberge ex Desm.) Verkley, Quaedvlieg & Crous, Sphaerulina frondicola (Fr.) Verkley, Quaedvlieg & Crous, Sphaerulina socia (Pass.) Quaedvlieg, Verkley & Crous; Epitypifications (basionyms) - Ascochyta lysimachiae Lib., Septoria astragali Roberge ex Desm., Septoria cerastii Roberge ex Desm., Septoria clematidis Roberge ex Desm., Septoria cruciatae Roberge ex Desm., Septoria spergulae Westend., Septoria epilobii Westend., Septoria galeopsidis Westend., Septoria gei Roberge ex Desm., Septoria hyperici Roberge ex Desm., Septoria rubi Westend., Septoria senecionis Westend., Septoria urticae Roberge ex Desm. PMID:24014901

Comparative and Evolutionary Analysis of the HES/HEY Gene Family Reveal Exon/Intron Loss and Teleost Specific Duplication Events

PubMed Central

Ma, Zhaowu; Zhou, Yang; Abbood, Nibras Najm; Liu, Jianfeng; Su, Li; Jia, Haibo; Guo, An-Yuan

2012-01-01

Background HES/HEY genes encode a family of basic helix-loop-helix (bHLH) transcription factors with both bHLH and Orange domain. HES/HEY proteins are direct targets of the Notch signaling pathway and play an essential role in developmental decisions, such as the developments of nervous system, somitogenesis, blood vessel and heart. Despite their important functions, the origin and evolution of this HES/HEY gene family has yet to be elucidated. Methods and Findings In this study, we identified genes of the HES/HEY family in representative species and performed evolutionary analysis to elucidate their origin and evolutionary process. Our results showed that the HES/HEY genes only existed in metazoans and may originate from the common ancestor of metazoans. We identified HES/HEY genes in more than 10 species representing the main lineages. Combining the bHLH and Orange domain sequences, we constructed the phylogenetic trees by different methods (Bayesian, ML, NJ and ME) and classified the HES/HEY gene family into four groups. Our results indicated that this gene family had undergone three expansions, which were along with the origins of Eumetazoa, vertebrate, and teleost. Gene structure analysis revealed that the HES/HEY genes were involved in exon and/or intron loss in different species lineages. Genes of this family were duplicated in bony fishes and doubled than other vertebrates. Furthermore, we studied the teleost-specific duplications in zebrafish and investigated the expression pattern of duplicated genes in different tissues by RT-PCR. Finally, we proposed a model to show the evolution of this gene family with processes of expansion, exon/intron loss, and motif loss. Conclusions Our study revealed the evolution of HES/HEY gene family, the expression and function divergence of duplicated genes, which also provide clues for the research of Notch function in development. This study shows a model of gene family analysis with gene structure evolution and duplication. PMID:22808219

Comparative and evolutionary analysis of the HES/HEY gene family reveal exon/intron loss and teleost specific duplication events.

PubMed

Zhou, Mi; Yan, Jun; Ma, Zhaowu; Zhou, Yang; Abbood, Nibras Najm; Liu, Jianfeng; Su, Li; Jia, Haibo; Guo, An-Yuan

2012-01-01

HES/HEY genes encode a family of basic helix-loop-helix (bHLH) transcription factors with both bHLH and Orange domain. HES/HEY proteins are direct targets of the Notch signaling pathway and play an essential role in developmental decisions, such as the developments of nervous system, somitogenesis, blood vessel and heart. Despite their important functions, the origin and evolution of this HES/HEY gene family has yet to be elucidated. In this study, we identified genes of the HES/HEY family in representative species and performed evolutionary analysis to elucidate their origin and evolutionary process. Our results showed that the HES/HEY genes only existed in metazoans and may originate from the common ancestor of metazoans. We identified HES/HEY genes in more than 10 species representing the main lineages. Combining the bHLH and Orange domain sequences, we constructed the phylogenetic trees by different methods (Bayesian, ML, NJ and ME) and classified the HES/HEY gene family into four groups. Our results indicated that this gene family had undergone three expansions, which were along with the origins of Eumetazoa, vertebrate, and teleost. Gene structure analysis revealed that the HES/HEY genes were involved in exon and/or intron loss in different species lineages. Genes of this family were duplicated in bony fishes and doubled than other vertebrates. Furthermore, we studied the teleost-specific duplications in zebrafish and investigated the expression pattern of duplicated genes in different tissues by RT-PCR. Finally, we proposed a model to show the evolution of this gene family with processes of expansion, exon/intron loss, and motif loss. Our study revealed the evolution of HES/HEY gene family, the expression and function divergence of duplicated genes, which also provide clues for the research of Notch function in development. This study shows a model of gene family analysis with gene structure evolution and duplication.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response in Arabidopsis[C][W

PubMed Central

Rigó, Gábor; Ayaydin, Ferhan; Tietz, Olaf; Zsigmond, Laura; Kovács, Hajnalka; Páy, Anikó; Salchert, Klaus; Darula, Zsuzsanna; Medzihradszky, Katalin F.; Szabados, László; Palme, Klaus; Koncz, Csaba; Cséplő, Ágnes

2013-01-01

CRK5 is a member of the Arabidopsis thaliana Ca2+/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5–green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane–associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling. PMID:23673979

Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.

PubMed

Holmes, Roger S; Wright, Matthew W; Laulederkind, Stanley J F; Cox, Laura A; Hosokawa, Masakiyo; Imai, Teruko; Ishibashi, Shun; Lehner, Richard; Miyazaki, Masao; Perkins, Everett J; Potter, Phillip M; Redinbo, Matthew R; Robert, Jacques; Satoh, Tetsuo; Yamashita, Tetsuro; Yan, Bingfan; Yokoi, Tsuyoshi; Zechner, Rudolf; Maltais, Lois J

2010-10-01

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

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

PubMed

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. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds.

PubMed

Duval, Frédéric D; Renard, Michelle; Jaquinod, Michel; Biou, Valérie; Montrichard, Françoise; Macherel, David

2002-11-01

Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix. CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5' and 3' untranslated regions. Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide. However, the three proteins activated in the same way the CaM-dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2.

PubMed

Hovey, Liam; Fowler, C Andrew; Mahling, Ryan; Lin, Zesen; Miller, Mark Stephen; Marx, Dagan C; Yoder, Jesse B; Kim, Elaine H; Tefft, Kristin M; Waite, Brett C; Feldkamp, Michael D; Yu, Liping; Shea, Madeline A

2017-05-01

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel Na V 1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat Na V 1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca 2+ ) 4 -CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca 2+ ) 4 -CaM, NMR demonstrated that Na V 1.2 IQ motif peptide (Na V 1.2 IQp ) exclusively made contacts with C-domain residues of CaM (CaM C ). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca 2+ ) 2 -CaM C bound to Na V 1.2 IQp . The polarity of (Ca 2+ ) 2 -CaM C relative to the IQ motif was opposite to that seen in apo CaM C -Na v 1.2 IQp (2KXW), revealing that CaM C recognizes nested, anti-parallel sites in Na v 1.2 IQp . Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaM N allowing interactions with non-IQ Na V 1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif. Copyright © 2017 Elsevier B.V. All rights reserved.

70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY*

PubMed Central

Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.

2013-01-01

We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156

The Role of Retrotransposons in Gene Family Expansions in the Human and Mouse Genomes

PubMed Central

Janoušek, Václav; Laukaitis, Christina M.; Yanchukov, Alexey

2016-01-01

Abstract Retrotransposons comprise a large portion of mammalian genomes. They contribute to structural changes and more importantly to gene regulation. The expansion and diversification of gene families have been implicated as sources of evolutionary novelties. Given the roles retrotransposons play in genomes, their contribution to the evolution of gene families warrants further exploration. In this study, we found a significant association between two major retrotransposon classes, LINEs and LTRs, and lineage-specific gene family expansions in both the human and mouse genomes. The distribution and diversity differ between LINEs and LTRs, suggesting that each has a distinct involvement in gene family expansion. LTRs are associated with open chromatin sites surrounding the gene families, supporting their involvement in gene regulation, whereas LINEs may play a structural role promoting gene duplication. Our findings also suggest that gene family expansions, especially in the mouse genome, undergo two phases. The first phase is characterized by elevated deposition of LTRs and their utilization in reshaping gene regulatory networks. The second phase is characterized by rapid gene family expansion due to continuous accumulation of LINEs and it appears that, in some instances at least, this could become a runaway process. We provide an example in which this has happened and we present a simulation supporting the possibility of the runaway process. Altogether we provide evidence of the contribution of retrotransposons to the expansion and evolution of gene families. Our findings emphasize the putative importance of these elements in diversification and adaptation in the human and mouse lineages. PMID:27503295

A Clinical and Molecular Genetic Study of 50 Families with Autosomal Recessive Parkinsonism Revealed Known and Novel Gene Mutations.

PubMed

Taghavi, Shaghayegh; Chaouni, Rita; Tafakhori, Abbas; Azcona, Luis J; Firouzabadi, Saghar Ghasemi; Omrani, Mir Davood; Jamshidi, Javad; Emamalizadeh, Babak; Shahidi, Gholam Ali; Ahmadi, Mona; Habibi, Seyed Amir Hassan; Ahmadifard, Azadeh; Fazeli, Atena; Motallebi, Marzieh; Petramfar, Peyman; Askarpour, Saeed; Askarpour, Shiva; Shahmohammadibeni, Hossein Ali; Shahmohammadibeni, Neda; Eftekhari, Hajar; Shafiei Zarneh, Amir Ehtesham; Mohammadihosseinabad, Saeed; Khorrami, Mehdi; Najmi, Safa; Chitsaz, Ahmad; Shokraeian, Parasto; Ehsanbakhsh, Hossein; Rezaeidian, Jalal; Ebrahimi Rad, Reza; Madadi, Faranak; Andarva, Monavvar; Alehabib, Elham; Atakhorrami, Minoo; Mortazavi, Seyed Erfan; Azimzadeh, Zahra; Bayat, Mahdis; Besharati, Amir Mohammad; Harati-Ghavi, Mohammad Ali; Omidvari, Samareh; Dehghani-Tafti, Zahra; Mohammadi, Faraz; Mohammad Hossein Pour, Banafsheh; Noorollahi Moghaddam, Hamid; Esmaili Shandiz, Ehsan; Habibi, Arman; Taherian-Esfahani, Zahra; Darvish, Hossein; Paisán-Ruiz, Coro

2018-04-01

In this study, the role of known Parkinson's disease (PD) genes was examined in families with autosomal recessive (AR) parkinsonism to assist with the differential diagnosis of PD. Some families without mutations in known genes were also subject to whole genome sequencing with the objective to identify novel parkinsonism-related genes. Families were selected from 4000 clinical files of patients with PD or parkinsonism. AR inheritance pattern, consanguinity, and a minimum of two affected individuals per family were used as inclusion criteria. For disease gene/mutation identification, multiplex ligation-dependent probe amplification, quantitative PCR, linkage, and Sanger and whole genome sequencing assays were carried out. A total of 116 patients (50 families) were examined. Fifty-four patients (46.55%; 22 families) were found to carry pathogenic mutations in known genes while a novel gene, not previously associated with parkinsonism, was found mutated in a single family (2 patients). Pathogenic mutations, including missense, nonsense, frameshift, and exon rearrangements, were found in Parkin, PINK1, DJ-1, SYNJ1, and VAC14 genes. In conclusion, variable phenotypic expressivity was seen across all families.

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato.

PubMed

Afiyanti, Mufidah; Chen, Hsien-Jung

2014-01-15

Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H2O2 homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl2, but not MgCl2 or CoCl2. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H2O2 level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl2 and SPCAM, and plays an important role in H2O2 homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band. Copyright © 2013 Elsevier GmbH. All rights reserved.

Eye and heart morphogenesis are dependent on melatonin signaling in chick embryos.

PubMed

Nogueira, Renato C; Sampaio, Lucia de Fatima S

2017-10-15

Calmodulin is vital for chick embryos morphogenesis in the incubation time 48-66 h when the rudimentary C-shaped heart attains an S-shaped pattern and the optic vesicles develop into optic cups. Melatonin is in the extraembryonic yolk sac of the avian egg; melatonin binds calmodulin. The aim of this study was to investigate the function of melatonin in the formation of the chick embryo optic cups and S-shaped heart, by pharmacological methods and immunoassays. Mel1a melatonin receptor immunofluorescence was distributed in the optic cups and rudimentary hearts. We separated embryonated chicken eggs at 48 h of incubation into basal, control and drug-treated groups, with treatment applied in the egg air sac. At 66 h of incubation, embryos were excised from the eggs and analyzed. Embryos from the basal, control (distilled water), melatonin and 6-chloromelatonin (melatonin receptor agonist) groups had regular optic cups and an S-shaped heart, while those from the calmidazolium (calmodulin inhibitor) group did not. Embryos from the luzindole (melatonin receptor antagonist) and prazosin (Mel1c melatonin receptor antagonist) groups did not have regular optic cups. Embryos from the 4-P-PDOT (Mel1b melatonin receptor antagonist) group did not have an S-shaped heart. Previous application of the melatonin, 6-chloromelatonin or forskolin (adenylate cyclase enhancer) prevented the abnormal appearance of chick embryos from the calmidazolium, luzindole, prazosin and 4-P-PDOT groups. However, 6-chloromelatonin and forskolin only partially prevented the development of defective eye cups in embryos from the calmidazolium group. The results suggested that melatonin modulates chick embryo morphogenesis via calmodulin and membrane receptors. © 2017. Published by The Company of Biologists Ltd.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

PubMed

Alkozi, Hanan Awad; Perez de Lara, María J; Pintor, Jesús

2017-09-01

Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishido, Hideki; Maruta, Shinsaku, E-mail: maruta@soka.ac.jp

Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesinmore » monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.« less

myo-Inositol 1,4,5-trisphosphate and Ca(2+)/calmodulin-dependent factors mediate transduction of compression-induced signals in bovine articular chondrocytes.

PubMed Central

Valhmu, Wilmot B; Raia, Frank J

2002-01-01

Although the effects of mechanical loading on chondrocyte metabolic activities have been extensively characterized, the sequence of events through which extracellular mechanical signals are transduced into chondrocytes and ultimately modulate cell activities is not well understood. Here, studies were performed to map out the sequential intracellular signalling pathways through which compression-induced signals modulate aggrecan mRNA levels in bovine articular chondrocytes. Bovine articular cartilage explants were subjected to a compressive stress of 0.1 MPa for 1 h in the presence or absence of inhibitors or antagonists of the phosphoinositol and Ca(2+)/calmodulin signalling pathways in order to determine the roles of second messengers and effector molecules of these pathways in transducing the compression-induced signals. In the absence of the inhibitors, aggrecan mRNA levels were stimulated by compression 2-4-fold relative to levels in tare-loaded (see below) explants. Treatment of the explants with graded levels of the protein kinase C inhibitor chelerythrine or bisindolylmaleimide I, followed by 1 h compressive loading, did not significantly alter the load-induced elevation of aggrecan mRNA levels. In contrast, thapsigargin, which depletes the Ins(1,4,5)P3-sensitive intracellular Ca(2+) stores, completely blocked the load response without significantly altering aggrecan mRNA levels in tare-loaded explants. Similarly, antagonists of the Ca(2+)/calmodulin signalling pathway dose-dependently or completely blocked the load-response. The results obtained demonstrate that transduction of the compression-induced aggrecan mRNA-regulating signals requires Ins(1,4,5)P3- and Ca(2+)/calmodulin-dependent signalling processes in bovine articular chondrocytes. PMID:11802800

Mapping Conformational Dynamics of Proteins Using Torsional Dynamics Simulations

PubMed Central

Gangupomu, Vamshi K.; Wagner, Jeffrey R.; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

2013-01-01

All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein’s experimentally established conformational substates. Conformational transition of calmodulin from the Ca2+-bound to the Ca2+-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins. PMID:23663843

Mapping conformational dynamics of proteins using torsional dynamics simulations.

PubMed

Gangupomu, Vamshi K; Wagner, Jeffrey R; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

2013-05-07

All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein's experimentally established conformational substates. Conformational transition of calmodulin from the Ca(2+)-bound to the Ca(2+)-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Hongliang; Hong, Da Hye; Kim, Han Sol

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 inactivationmore » 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.« less

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

PubMed

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 (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick 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.

Evaluation of Gene-Based Family-Based Methods to Detect Novel Genes Associated With Familial Late Onset Alzheimer Disease

PubMed Central

Fernández, Maria V.; Budde, John; Del-Aguila, Jorge L.; Ibañez, Laura; Deming, Yuetiva; Harari, Oscar; Norton, Joanne; Morris, John C.; Goate, Alison M.; Cruchaga, Carlos

2018-01-01

Gene-based tests to study the combined effect of rare variants on a particular phenotype have been widely developed for case-control studies, but their evolution and adaptation for family-based studies, especially studies of complex incomplete families, has been slower. In this study, we have performed a practical examination of all the latest gene-based methods available for family-based study designs using both simulated and real datasets. We examined the performance of several collapsing, variance-component, and transmission disequilibrium tests across eight different software packages and 22 models utilizing a cohort of 285 families (N = 1,235) with late-onset Alzheimer disease (LOAD). After a thorough examination of each of these tests, we propose a methodological approach to identify, with high confidence, genes associated with the tested phenotype and we provide recommendations to select the best software and model for family-based gene-based analyses. Additionally, in our dataset, we identified PTK2B, a GWAS candidate gene for sporadic AD, along with six novel genes (CHRD, CLCN2, HDLBP, CPAMD8, NLRP9, and MAS1L) as candidate genes for familial LOAD. PMID:29670507

Evaluation of Gene-Based Family-Based Methods to Detect Novel Genes Associated With Familial Late Onset Alzheimer Disease.

PubMed

Fernández, Maria V; Budde, John; Del-Aguila, Jorge L; Ibañez, Laura; Deming, Yuetiva; Harari, Oscar; Norton, Joanne; Morris, John C; Goate, Alison M; Cruchaga, Carlos

2018-01-01

Gene-based tests to study the combined effect of rare variants on a particular phenotype have been widely developed for case-control studies, but their evolution and adaptation for family-based studies, especially studies of complex incomplete families, has been slower. In this study, we have performed a practical examination of all the latest gene-based methods available for family-based study designs using both simulated and real datasets. We examined the performance of several collapsing, variance-component, and transmission disequilibrium tests across eight different software packages and 22 models utilizing a cohort of 285 families ( N = 1,235) with late-onset Alzheimer disease (LOAD). After a thorough examination of each of these tests, we propose a methodological approach to identify, with high confidence, genes associated with the tested phenotype and we provide recommendations to select the best software and model for family-based gene-based analyses. Additionally, in our dataset, we identified PTK2B , a GWAS candidate gene for sporadic AD, along with six novel genes ( CHRD, CLCN2, HDLBP, CPAMD8, NLRP9 , and MAS1L ) as candidate genes for familial LOAD.

A Plant Phytosulfokine Peptide Initiates Auxin-Dependent Immunity through Cytosolic Ca2+ Signaling in Tomato[OPEN

PubMed Central

Zhang, Huan; Hu, Zhangjian; Lei, Cui; Zheng, Chenfei; Wang, Jiao; Shao, Shujun; Li, Xin; Xia, Xiaojian; Cai, Xinzhong

2018-01-01

Phytosulfokine (PSK) is a disulfated pentapeptide that is an important signaling molecule. Although it has recently been implicated in plant defenses to pathogen infection, the mechanisms involved remain poorly understood. Using surface plasmon resonance and gene silencing approaches, we showed that the tomato (Solanum lycopersicum) PSK receptor PSKR1, rather than PSKR2, functioned as the major PSK receptor in immune responses. Silencing of PSK signaling genes rendered tomato more susceptible to infection by the economically important necrotrophic pathogen Botrytis cinerea. Analysis of tomato mutants defective in either defense hormone biosynthesis or signaling demonstrated that PSK-induced immunity required auxin biosynthesis and associated defense pathways. Here, using aequorin-expressing tomato plants, we provide evidence that PSK perception by tomato PSKR1 elevated cytosolic [Ca2+], leading to auxin-dependent immune responses via enhanced binding activity between calmodulins and the auxin biosynthetic YUCs. Thus, our data demonstrate that PSK acts as a damage-associated molecular pattern and is perceived mainly by PSKR1, which increases cytosolic [Ca2+] and activates auxin-mediated pathways that enhance immunity of tomato plants to B. cinerea. PMID:29511053

Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

PubMed Central

Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

2015-01-01

Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

PubMed

Rauhala, Leena; Hämäläinen, Lasse; Dunlop, Thomas W; Pehkonen, Petri; Bart, Geneviève; Kokkonen, Maarit; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

2015-12-25

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Penicillium simile sp. nov. revealed by morphological and phylogenetic analysis.

PubMed

Davolos, Domenico; Pietrangeli, Biancamaria; Persiani, Anna Maria; Maggi, Oriana

2012-02-01

The morphology of three phenetically identical Penicillium isolates, collected from the bioaerosol in a restoration laboratory in Italy, displayed macro- and microscopic characteristics that were similar though not completely ascribable to Penicillium raistrickii. For this reason, a phylogenetic approach based on DNA sequencing analysis was performed to establish both the taxonomic status and the evolutionary relationships of these three peculiar isolates in relation to previously described species of the genus Penicillium. We used four nuclear loci (both rRNA and protein coding genes) that have previously proved useful for the molecular investigation of taxa belonging to the genus Penicillium at various evolutionary levels. The internal transcribed spacer region (ITS1-5.8S-ITS2), domains D1 and D2 of the 28S rDNA, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and sequenced. Analysis of the rRNA genes and of the benA and cmd sequence data indicates the presence of three isogenic isolates belonging to a genetically distinct species of the genus Penicillium, here described and named Penicillium simile sp. nov. (ATCC MYA-4591(T)  = CBS 129191(T)). This novel species is phylogenetically different from P. raistrickii and other related species of the genus Penicillium (e.g. Penicillium scabrosum), from which it can be distinguished on the basis of morphological trait analysis.

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

PubMed

Mogollon, Catherin Marin; van Pul, Fiona J A; Imai, Takashi; Ramesar, Jai; Chevalley-Maurel, Séverine; de Roo, Guido M; Veld, Sabrina A J; Kroeze, Hans; Franke-Fayard, Blandine M D; Janse, Chris J; Khan, Shahid M

2016-01-01

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

PubMed Central

Mogollon, Catherin Marin; van Pul, Fiona J. A.; Imai, Takashi; Ramesar, Jai; Chevalley-Maurel, Séverine; de Roo, Guido M.; Veld, Sabrina A. J.; Kroeze, Hans; Franke-Fayard, Blandine M. D.; Janse, Chris J.

2016-01-01

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters. PMID:27997583

Mutations in TULP1, NR2E3, and MFRP genes in Indian families with autosomal recessive retinitis pigmentosa

PubMed Central

Singh, Hardeep; Sahini, Nishika; Jalali, Subhadra; Mohan, Gayathri

2012-01-01

Purpose To identify genes underlying autosomal recessive retinitis pigmentosa (ARRP) by homozygosity mapping. Methods Families with ARRP were recruited after complete ophthalmic evaluation of all members and diagnosis of RP by predefined criteria. Genomic DNA from affected members of 26 families was genotyped on Illumina single nucleotide polymorphism (SNP) 6.0 K arrays with standard procedures. Genotypes were evaluated for homozygous regions that were common and concordant between affected members of each family. The genes mapping to homozygous intervals within these families were screened for pathogenic changes with PCR amplification and sequencing of coding regions. Cosegegration of sequence changes with disease was determined within each pedigree, and each variation was tested for presence in 100 unrelated normal controls. Results A genome-wide scan for homozygosity showed homozygous regions harboring the tubby like protein 1 gene (TULP1; chromosome 6) in one family, the nuclear receptor subfamily 2, group E, member 3 gene (NR2E3; chromosome 15) in three families, and the membrane frizzled-related protein gene (MFRP; chromosome 11) in one family. Screening of the three genes in the respective families revealed homozygous disease-causing mutations in three families. These included a missense mutation in TULP1, a deletion-cum-insertion in NR2E3, and a single base deletion in MFRP. Patients from all three families had a rod-cone type of dystrophy with night blindness initially. The NR2E3 and MFRP genes were associated with fundus features atypical of RP. Conclusions This study shows involvement of the TULP1, NR2E3, and MFRP genes in ARRP in Indian cases. Genome-wide screening with SNP arrays followed by a prioritized candidate gene evaluation is useful in identifying genes in these patients. PMID:22605927

Genome-wide coexpression dynamics: Theory and application

PubMed Central

Li, Ker-Chau

2002-01-01

High-throughput expression profiling enables the global study of gene activities. Genes with positively correlated expression profiles are likely to encode functionally related proteins. However, all biological processes are interlocked, and each protein may play multiple cellular roles. Thus the coexpression of any two functionally related genes may depend on the constantly varying, yet often-unknown cellular state. To initiate a systematic study on this issue, a theory of coexpression dynamics is presented. This theory is used to rationalize a strategy of conducting a genome-wide search for the most critical cellular players that may affect the coexpression pattern of any two genes. In one example, using a yeast data set, our method reveals how the enzymes associated with the urea cycle are expressed to ensure proper mass flow of the involved metabolites. The correlation between ARG2 and CAR2 is found to change from positive to negative as the expression level of CPA2 increases. This delicate interplay in correlation signifies a remarkable control on the influx and efflux of ornithine and reflects well the intrinsic cellular demand for arginine. In addition to the urea cycle, our examples include SCH9 and CYR1 (both implicated in a recent longevity study), cytochrome c1 (mitochondrial electron transport), calmodulin (main calcium-binding protein), PFK1 and PFK2 (glycolysis), and two genes, ECM1 and YNL101W, the functions of which are newly revealed. The complexity in computation is eased by a new result from mathematical statistics. PMID:12486219

Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation.

PubMed

Zhang, Chun Li; McKinsey, Timothy A; Olson, Eric N

2002-10-01

Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscle differentiation through associations with the myocyte enhancer factor 2 (MEF2) transcription factor. MEF2-interacting transcription repressor (MITR) is an amino-terminal splice variant of HDAC9 that also potently inhibits MEF2 transcriptional activity despite lacking a catalytic domain. Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatin protein 1 (HP1), an adaptor protein that recognizes methylated lysines within histone tails and mediates transcriptional repression by recruiting histone methyltransferase. Promyogenic signals provided by calcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITR and HDACs with HP1. Since the histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs, the interaction of MITR and HDACs with HP1 provides an efficient mechanism for silencing MEF2 target genes by coupling histone deacetylation and methylation. Indeed, nucleosomal histones surrounding a MEF2-binding site in the myogenin gene promoter are highly methylated in undifferentiated myoblasts, when the gene is silent, and become acetylated during muscle differentiation, when the myogenin gene is expressed at high levels. The ability of MEF2 to recruit a histone methyltransferase to target gene promoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling to disrupt these interactions provides an efficient mechanism for signal-dependent regulation of the epigenetic events controlling muscle differentiation.

Association of Class II Histone Deacetylases with Heterochromatin Protein 1: Potential Role for Histone Methylation in Control of Muscle Differentiation

PubMed Central

Zhang, Chun Li; McKinsey, Timothy A.; Olson, Eric N.

2002-01-01

Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscle differentiation through associations with the myocyte enhancer factor 2 (MEF2) transcription factor. MEF2-interacting transcription repressor (MITR) is an amino-terminal splice variant of HDAC9 that also potently inhibits MEF2 transcriptional activity despite lacking a catalytic domain. Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatin protein 1 (HP1), an adaptor protein that recognizes methylated lysines within histone tails and mediates transcriptional repression by recruiting histone methyltransferase. Promyogenic signals provided by calcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITR and HDACs with HP1. Since the histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs, the interaction of MITR and HDACs with HP1 provides an efficient mechanism for silencing MEF2 target genes by coupling histone deacetylation and methylation. Indeed, nucleosomal histones surrounding a MEF2-binding site in the myogenin gene promoter are highly methylated in undifferentiated myoblasts, when the gene is silent, and become acetylated during muscle differentiation, when the myogenin gene is expressed at high levels. The ability of MEF2 to recruit a histone methyltransferase to target gene promoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling to disrupt these interactions provides an efficient mechanism for signal-dependent regulation of the epigenetic events controlling muscle differentiation. PMID:12242305

Apoptotic activity and gene responses in Drosophila melanogaster S2 cells, induced by azadirachtin A.

PubMed

Xu, Lin; Li, Sheng; Ran, Xueqin; Liu, Chang; Lin, Rutao; Wang, Jiafu

2016-09-01

Azadirachtin has been used as an antifeedant and growth disruption agent for many insect species. Previous investigations have reported the apoptotic effects of azadirachtin on some insect cells, but the molecular mechanisms are still not clear. This study investigated the underlying molecular mechanisms for the apoptotic effects induced by azadirachtin on Drosophila melanogaster S2 cells in vitro. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that azadirachtin exhibited significant cytotoxicity to S2 cells in a time- and dose-dependent manner. The changes in cellular morphology and the DNA fragmentation demonstrated that azadirachtin induced remarkable apoptosis of S2 cells. Expression levels of 276 genes were found to be significantly changed in S2 cells after exposure to azadirachtin, as detected by Drosophila genome array. Among these genes, calmodulin (CaM) was the most highly upregulated gene. Azadirachtin was further demonstrated to trigger intracellular Ca(2+) release in S2 cells. The genes related to the apoptosis pathway, determined from chip data, were validated by the real-time quantitative polymerase chain reaction method. The results showed that azadirachtin-mediated intracellular Ca(2+) release was the primary event that triggered apoptosis in Drosophila S2 cells through both pathways of the Ca(2+) -CaM and EcR/Usp signalling cascade. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Disbalance of calcium regulation-related genes in broiler hearts induced by selenium deficiency.

PubMed

Zhang, Ziwei; Liu, Man; Guan, Zhenqiong; Yang, Jie; Liu, Zhonghua; Xu, Shiwen

2017-06-01

Dietary selenium (Se) deficiency may influence the calcium (Ca) homeostasis in broilers. Our objective was to investigate the effects of Se deficiency on Ca regulation-related genes in broiler hearts. In the present study, 1-day-old broilers were fed either a commercial diet (as control group) with 0.15 mg/kg Se or a Se-deficient diet (as L group) with 0.033 mg/kg Se for 35 days. We examined the mRNA expression levels of 15 Ca regulation-related genes (ITPR 1, ITPR 2, ITPR3, RyR2, RyR3, SERCA1s, SLC8A1, PMCA1, CACNA1S, TRPC1, TRPC3, stromal interacting molecule 1, ORAI1, calmodulin (CaLM) and calreticulin (CRT) in broiler hearts. Then, Kyoto Encyclopedia of Genes and Genomes analysis, protein-protein interactions (PPI) analysis and correlation analysis were performed to analyse the relationships between these genes. The results showed that the mRNA expression levels of ITPR 1, ITPR 2, RyR2, RyR3, SERCA1s, SLC8A1, PMCA1, CACNA1S, CaLM and CRT were generally decreased by Se deficiency, while mRNA expression levels of TRPC1, TRPC3, stromal interacting molecule 1, ORAI1 and ITPR3 were increased by Se deficiency. Kyoto Encyclopedia of Genes and Genomes and PPI analysis showed that these Ca regulation-related genes are involved in the Ca signalling pathway and a total of 15 PPIs with a combined score of >0.4 were obtained. In conclusion, the results demonstrated that Se deficiency might cause heart injury via modulating the Ca-related pathway genes, and then induce Ca 2+ overload in the heart of broilers.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

PubMed

Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

PubMed Central

Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

PubMed Central

Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

2015-01-01

Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

Identification of a novel Gig2 gene family specific to non-amniote vertebrates.

PubMed

Zhang, Yi-Bing; Liu, Ting-Kai; Jiang, Jun; Shi, Jun; Liu, Ying; Li, Shun; Gui, Jian-Fang

2013-01-01

Gig2 (grass carp reovirus (GCRV)-induced gene 2) is first identified as a novel fish interferon (IFN)-stimulated gene (ISG). Overexpression of a zebrafish Gig2 gene can protect cultured fish cells from virus infection. In the present study, we identify a novel gene family that is comprised of genes homologous to the previously characterized Gig2. EST/GSS search and in silico cloning identify 190 Gig2 homologous genes in 51 vertebrate species ranged from lampreys to amphibians. Further large-scale search of vertebrate and invertebrate genome databases indicate that Gig2 gene family is specific to non-amniotes including lampreys, sharks/rays, ray-finned fishes and amphibians. Phylogenetic analysis and synteny analysis reveal lineage-specific expansion of Gig2 gene family and also provide valuable evidence for the fish-specific genome duplication (FSGD) hypothesis. Although Gig2 family proteins exhibit no significant sequence similarity to any known proteins, a typical Gig2 protein appears to consist of two conserved parts: an N-terminus that bears very low homology to the catalytic domains of poly(ADP-ribose) polymerases (PARPs), and a novel C-terminal domain that is unique to this gene family. Expression profiling of zebrafish Gig2 family genes shows that some duplicate pairs have diverged in function via acquisition of novel spatial and/or temporal expression under stresses. The specificity of this gene family to non-amniotes might contribute to a large extent to distinct physiology in non-amniote vertebrates.

The cumulative analgesic effect of repeated electroacupuncture involves synaptic remodeling in the hippocampal CA3 region☆

PubMed Central

Xu, Qiuling; Liu, Tao; Chen, Shuping; Gao, Yonghui; Wang, Junying; Qiao, Lina; Liu, Junling

2012-01-01

In the present study, we examined the analgesic effect of repeated electroacupuncture at bilateral Zusanli (ST36) and Yanglingquan (GB34) once a day for 14 consecutive days in a rat model of chronic sciatic nerve constriction injury-induced neuropathic pain. In addition, concomitant changes in calcium/calmodulin-dependent protein kinase II expression and synaptic ultrastructure of neurons in the hippocampal CA3 region were examined. The thermal pain threshold (paw withdrawal latency) was increased significantly in both groups at 2 weeks after electroacupuncture intervention compared with 2 days of electroacupuncture. In ovariectomized rats with chronic constriction injury, the analgesic effect was significantly reduced. Electroacupuncture for 2 weeks significantly diminished the injury-induced increase in synaptic cleft width and thinning of the postsynaptic density, and it significantly suppressed the down-regulation of intracellular calcium/calmodulin-dependent protein kinase II expression in the hippocampal CA3 region. Repeated electroacupuncture intervention had a cumulative analgesic effect on injury-induced neuropathic pain reactions, and it led to synaptic remodeling of hippocampal neurons and upregulated calcium/calmodulin-dependent protein kinase II expression in the hippocampal CA3 region. PMID:25657670

The Arrhythmogenic Calmodulin Mutation D129G Dysregulates Cell Growth, Calmodulin-dependent Kinase II Activity, and Cardiac Function in Zebrafish*

PubMed Central

Zacharias, Triantafyllos; Kulej, Katarzyna; Wang, Kevin; Torggler, Raffaela; la Cour, Jonas M.

2016-01-01

Calmodulin (CaM) is a Ca2+ binding protein modulating multiple targets, several of which are associated with cardiac pathophysiology. Recently, CaM mutations were linked to heart arrhythmia. CaM is crucial for cell growth and viability, yet the effect of the arrhythmogenic CaM mutations on cell viability, as well as heart rhythm, remains unknown, and only a few targets with relevance for heart physiology have been analyzed for their response to mutant CaM. We show that the arrhythmia-associated CaM mutants support growth and viability of DT40 cells in the absence of WT CaM except for the long QT syndrome mutant CaM D129G. Of the six CaM mutants tested (N53I, F89L, D95V, N97S, D129G, and F141L), three showed a decreased activation of Ca2+/CaM-dependent kinase II, most prominently the D129G CaM mutation, which was incapable of stimulating Thr286 autophosphorylation. Furthermore, the CaM D129G mutation led to bradycardia in zebrafish and an arrhythmic phenotype in a subset of the analyzed zebrafish. PMID:27815504

Structure-Activity Relationships for the Antifungal Activity of Selective Estrogen Receptor Antagonists Related to Tamoxifen

PubMed Central

Butts, Arielle; Martin, Jennifer A.; DiDone, Louis; Bradley, Erin K.; Mutz, Mitchell; Krysan, Damian J.

2015-01-01

Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold. PMID:26016941

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodríguez Guilbe, María M.; Protein Research and Development Center, University of Puerto Rico; Alfaro Malavé, Elisa C.

The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, thismore » protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.« less

Activation of the edema factor of Bacillus anthracis by calmodulin: evidence of an interplay between the EF-calmodulin interaction and calcium binding.

PubMed

Laine, Elodie; Martínez, Leandro; Blondel, Arnaud; Malliavin, Thérèse E

2010-10-06

Calmodulin (CaM) is a remarkably flexible protein which can bind multiple targets in response to changes in intracellular calcium concentration. It contains four calcium-binding sites, arranged in two globular domains. The calcium affinity of CaM N-terminal domain (N-CaM) is dramatically reduced when the complex with the edema factor (EF) of Bacillus anthracis is formed. Here, an atomic explanation for this reduced affinity is proposed through molecular dynamics simulations and free energy perturbation calculations of the EF-CaM complex starting from different crystallographic models. The simulations show that electrostatic interactions between CaM and EF disfavor the opening of N-CaM domains usually induced by calcium binding. Relative calcium affinities of the N-CaM binding sites are probed by free energy perturbation, and dissociation probabilities are evaluated with locally enhanced sampling simulations. We show that EF impairs calcium binding on N-CaM through a direct conformational restraint on Site 1, by an indirect destabilization of Site 2, and by reducing the cooperativity between the two sites. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin.

PubMed

Floris, Federico; Chiron, Lionel; Lynch, Alice M; Barrow, Mark P; Delsuc, Marc-André; O'Connor, Peter B

2018-06-04

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Graphical Abstract Two-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin.

VH gene family expression in mice with the xid defect

PubMed Central

1991-01-01

Preferential use of particular VH gene families in the response to specific antigens has been demonstrated in several systems. The lack of responses to certain types of antigens, therefore, could be the result of deletion of or failure to express some VH genes. Because CBA/N mice, which carry the X-linked immunodeficiency (xid) gene defect, have been shown to be unresponsive to thymus-independent polysaccharide antigens, it was of interest to examine if this unresponsiveness could be accounted for by abnormal expression of particular VH gene families. Using in situ hybridization on B cell colonies, we determined the expression of nine VH gene families in CBA/CaHN females (genotypically normal), CBA/N males (xid) and females (xid), and (CBA/N x CBA/CaHN)F1 males (xid) and females (phenotypically normal). Our results indicate that VH gene family expression, including the S107 family, in CBA/N males and F1 males, is similar to that of CBA/CaHN and F1 females with predominant expression of J558, the largest gene family, in all individuals. Interestingly, CBA/N female mice, which carry two defective X chromosomes, as a group expressed significantly reduced levels of the J558 gene family, and as individuals showed variation in which family was predominantly expressed. We conclude that the unresponsiveness of mice with the xid defect to polysaccharide antigens can not attributed to a failure to express the nine VH gene families that we examined. Our findings do not support previous studies (Primi, D., and P.-A. Cazenave 1986. J. Exp. Med. 165:357), which found an absence of expression of the S107 family in xid mice. PMID:1711566

Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes.

PubMed

Cabañes, F J; Bragulat, M R; Castellá, G

2013-12-01

Aspergillus carbonarius is the main responsible source of ochratoxin A (OTA) in food commodities such as wine, grapes or dried vine fruits from main viticultural regions worldwide. Besides, OTA production is a very consistent property of this species and for this reason atoxigenic isolates of A. carbonarius are very rarely found in natural environments. In the present study, for the first time, three nonochratoxigenic wild strains of A. carbonarius have been discovered, unambiguously identified, characterized in deep and compared to ochratoxigenic strains of the same species. In addition, polyketide synthase (pks) genes suggested to be involved in OTA biosynthesis were also screened in these strains. The identification of the strains was confirmed by ITS-5.8S rRNA, β-tubulin and calmodulin gene sequencing. The three atoxigenic strains did not produce OTA in a conducive culture medium at any of the temperatures and times of incubation tested. Five ketosynthase domains from pks genes previously described in A. carbonarius were detected both in ochratoxigenic and in nonochratoxigenic strains. Atoxigenic strains of A. carbonarius could be useful as biotechnological agents to be used in food industry and as biological agents for control of OTA production in vineyards and other crops. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sexual reproduction as the cause of heat resistance in the food spoilage fungus Byssochlamys spectabilis (anamorph Paecilomyces variotii).

PubMed

Houbraken, Jos; Varga, János; Rico-Munoz, Emilia; Johnson, Shawn; Samson, Robert A

2008-03-01

Paecilomyces variotii is a common cosmopolitan species that is able to spoil various food- and feedstuffs and is frequently encountered in heat-treated products. However, isolates from heat-treated products rarely form ascospores. In this study we examined by using molecular techniques and mating tests whether this species can undergo a sexual cycle and form ascospores. The population structure of this species was examined by analyzing the nuclear ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and the 5.8S rRNA gene, as well as partial beta-tubulin, actin, and calmodulin gene sequences. Phylogenetic analyses revealed that P. variotii is a highly variable species. Partition homogeneity tests revealed that P. variotii has a recombining population structure. In addition to sequence analyses, mating experiments indicated that P. variotii is able to form ascomata and ascospores in culture in a heterothallic manner. The distribution of MAT1-1 and MAT1-2 genes showed a 1:1 ratio in the progeny of the mating experiments. From the sequence analyses and mating data we conclude that P. variotii is the anamorph of Talaromyces spectabilis and that it has a biallelic heterothallic mating system. Since Paecilomyces sensu stricto anamorphs group within Byssochlamys, a new combination Byssochlamys spectabilis is proposed.

Effects of Gene Duplication, Positive Selection, and Shifts in Gene Expression on the Evolution of the Venom Gland Transcriptome in Widow Spiders

PubMed Central

Haney, Robert A.; Clarke, Thomas H.; Gadgil, Rujuta; Fitzpatrick, Ryan; Hayashi, Cheryl Y.; Ayoub, Nadia A.; Garb, Jessica E.

2016-01-01

Gene duplication and positive selection can be important determinants of the evolution of venom, a protein-rich secretion used in prey capture and defense. In a typical model of venom evolution, gene duplicates switch to venom gland expression and change function under the action of positive selection, which together with further duplication produces large gene families encoding diverse toxins. Although these processes have been demonstrated for individual toxin families, high-throughput multitissue sequencing of closely related venomous species can provide insights into evolutionary dynamics at the scale of the entire venom gland transcriptome. By assembling and analyzing multitissue transcriptomes from the Western black widow spider and two closely related species with distinct venom toxicity phenotypes, we do not find that gene duplication and duplicate retention is greater in gene families with venom gland biased expression in comparison with broadly expressed families. Positive selection has acted on some venom toxin families, but does not appear to be in excess for families with venom gland biased expression. Moreover, we find 309 distinct gene families that have single transcripts with venom gland biased expression, suggesting that the switching of genes to venom gland expression in numerous unrelated gene families has been a dominant mode of evolution. We also find ample variation in protein sequences of venom gland–specific transcripts, lineage-specific family sizes, and ortholog expression among species. This variation might contribute to the variable venom toxicity of these species. PMID:26733576

Genome Dynamics Explain the Evolution of Flowering Time CCT Domain Gene Families in the Poaceae

PubMed Central

Cockram, James; Thiel, Thomas; Steuernagel, Burkhard; Stein, Nils; Taudien, Stefan; Bailey, Paul C.; O'Sullivan, Donal M.

2012-01-01

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken. PMID:23028921

Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae.

PubMed

Cockram, James; Thiel, Thomas; Steuernagel, Burkhard; Stein, Nils; Taudien, Stefan; Bailey, Paul C; O'Sullivan, Donal M

2012-01-01

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.

Molecular Mechanisms for the Coupling of Endocytosis to Exocytosis in Neurons

PubMed Central

Xie, Zhenli; Long, Jiangang; Liu, Jiankang; Chai, Zuying; Kang, Xinjiang; Wang, Changhe

2017-01-01

Neuronal communication and brain function mainly depend on the fundamental biological events of neurotransmission, including the exocytosis of presynaptic vesicles (SVs) for neurotransmitter release and the subsequent endocytosis for SV retrieval. Neurotransmitters are released through the Ca2+- and SNARE-dependent fusion of SVs with the presynaptic plasma membrane. Following exocytosis, endocytosis occurs immediately to retrieve SV membrane and fusion machinery for local recycling and thus maintain the homeostasis of synaptic structure and sustained neurotransmission. Apart from the general endocytic machinery, recent studies have also revealed the involvement of SNARE proteins (synaptobrevin, SNAP25 and syntaxin), synaptophysin, Ca2+/calmodulin, and members of the synaptotagmin protein family (Syt1, Syt4, Syt7 and Syt11) in the balance and tight coupling of exo-endocytosis in neurons. Here, we provide an overview of recent progress in understanding how these neuron-specific adaptors coordinate to ensure precise and efficient endocytosis during neurotransmission. PMID:28348516

Cocaine- and amphetamine-regulated transcript peptide in the nucleus accumbens shell inhibits cocaine-induced locomotor sensitization to transient over-expression of α-Ca2+ /calmodulin-dependent protein kinase II.

PubMed

Xiong, Lixia; Meng, Qing; Sun, Xi; Lu, Xiangtong; Fu, Qiang; Peng, Qinghua; Yang, Jianhua; Oh, Ki-Wan; Hu, Zhenzhen

2018-01-04

Cocaine- and amphetamine-regulated transcript (CART) peptide is a widely distributed neurotransmitter that attenuates cocaine-induced locomotor activity when injected into the nucleus accumbens (NAc). Our previous work first confirmed that the inhibitory mechanism of the CART peptide on cocaine-induced locomotor activity is related to a reduction in cocaine-enhanced phosphorylated Ca 2+ /calmodulin-dependent protein kinaseIIα (pCaMKIIα) and the enhancement of cocaine-induced D3R function. This study investigated whether CART peptide inhibited cocaine-induced locomotor activity via inhibition of interactions between pCaMKIIα and the D3 dopamine receptor (D3R). We demonstrated that lentivirus-mediated gene transfer transiently increased pCaMKIIα expression, which peaked at 10 days after microinjection into the rat NAc shell, and induced a significant increase in Ca 2+ influx along with greater behavioral sensitivity in the open field test after intraperitoneal injections of cocaine (15 mg/kg). However, western blot analysis and coimmunoprecipitation demonstrated that CART peptide treatment in lentivirus-transfected CaMKIIα-over-expressing NAc rat tissues or cells prior to cocaine administration inhibited the cocaine-induced Ca 2+ influx and attenuated the cocaine-increased pCaMKIIα expression in lentivirus-transfected CaMKIIα-over-expressing cells. CART peptide decreased the cocaine-enhanced phosphorylated cAMP response element binding protein (pCREB) expression via inhibition of the pCaMKIIα-D3R interaction, which may account for the prolonged locomotor sensitization induced by repeated cocaine treatment in lentivirus-transfected CaMKIIα-over-expressing cells. These results provide strong evidence for the inhibitory modulation of CART peptide in cocaine-induced locomotor sensitization. © 2018 International Society for Neurochemistry.

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

NASA Technical Reports Server (NTRS)

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.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages.

PubMed

Datta, Debika; Khatri, Preeti; Banerjee, Chaitali; Singh, Ambika; Meena, Ramavatar; Saha, Dhira Rani; Raman, Rajagopal; Rajamani, Paulraj; Mitra, Abhijit; Mazumder, Shibnath

2016-01-01

Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

Genome-Wide Identification, Characterization and Expression Analysis of the Chalcone Synthase Family in Maize

PubMed Central

Han, Yahui; Ding, Ting; Su, Bo; Jiang, Haiyang

2016-01-01

Members of the chalcone synthase (CHS) family participate in the synthesis of a series of secondary metabolites in plants, fungi and bacteria. The metabolites play important roles in protecting land plants against various environmental stresses during the evolutionary process. Our research was conducted on comprehensive investigation of CHS genes in maize (Zea mays L.), including their phylogenetic relationships, gene structures, chromosomal locations and expression analysis. Fourteen CHS genes (ZmCHS01–14) were identified in the genome of maize, representing one of the largest numbers of CHS family members identified in one organism to date. The gene family was classified into four major classes (classes I–IV) based on their phylogenetic relationships. Most of them contained two exons and one intron. The 14 genes were unevenly located on six chromosomes. Two segmental duplication events were identified, which might contribute to the expansion of the maize CHS gene family to some extent. In addition, quantitative real-time PCR and microarray data analyses suggested that ZmCHS genes exhibited various expression patterns, indicating functional diversification of the ZmCHS genes. Our results will contribute to future studies of the complexity of the CHS gene family in maize and provide valuable information for the systematic analysis of the functions of the CHS gene family. PMID:26828478

Quantifying the major mechanisms of recent gene duplications in the human and mouse genomes: a novel strategy to estimate gene duplication rates

PubMed Central

Pan, Deng; Zhang, Liqing

2007-01-01

Background The rate of gene duplication is an important parameter in the study of evolution, but the influence of gene conversion and technical problems have confounded previous attempts to provide a satisfying estimate. We propose a new strategy to estimate the rate that involves separate quantification of the rates of two different mechanisms of gene duplication and subsequent combination of the two rates, based on their respective contributions to the overall gene duplication rate. Results Previous estimates of gene duplication rates are based on small gene families. Therefore, to assess the applicability of this to families of all sizes, we looked at both two-copy gene families and the entire genome. We studied unequal crossover and retrotransposition, and found that these mechanisms of gene duplication are largely independent and account for a substantial amount of duplicated genes. Unequal crossover contributed more to duplications in the entire genome than retrotransposition did, but this contribution was significantly less in two-copy gene families, and duplicated genes arising from this mechanism are more likely to be retained. Combining rates of duplication using the two mechanisms, we estimated the overall rates to be from approximately 0.515 to 1.49 × 10-3 per gene per million years in human, and from approximately 1.23 to 4.23 × 10-3 in mouse. The rates estimated from two-copy gene families are always lower than those from the entire genome, and so it is not appropriate to use small families to estimate the rate for the entire genome. Conclusion We present a novel strategy for estimating gene duplication rates. Our results show that different mechanisms contribute differently to the evolution of small and large gene families. PMID:17683522

Identification and expression profiling analysis of TCP family genes involved in growth and development in maize.

PubMed

Chai, Wenbo; Jiang, Pengfei; Huang, Guoyu; Jiang, Haiyang; Li, Xiaoyu

2017-10-01

The TCP family is a group of plant-specific transcription factors. TCP genes encode proteins harboring bHLH structure, which is implicated in DNA binding and protein-protein interactions and known as the TCP domain. TCP genes play important roles in plant development and have been evolutionarily and functionally elaborated in various plants, however, no overall phylogenetic analysis or expression profiling of TCP genes in Zea mays has been reported. In the present study, a systematic analysis of molecular evolution and functional prediction of TCP family genes in maize ( Z . mays L.) has been conducted. We performed a genome-wide survey of TCP genes in maize, revealing the gene structure, chromosomal location and phylogenetic relationship of family members. Microsynteny between grass species and tissue-specific expression profiles were also investigated. In total, 29 TCP genes were identified in the maize genome, unevenly distributed on the 10 maize chromosomes. Additionally, ZmTCP genes were categorized into nine classes based on phylogeny and purifying selection may largely be responsible for maintaining the functions of maize TCP genes. What's more, microsynteny analysis suggested that TCP genes have been conserved during evolution. Finally, expression analysis revealed that most TCP genes are expressed in the stem and ear, which suggests that ZmTCP genes influence stem and ear growth. This result is consistent with the previous finding that maize TCP genes represses the growth of axillary organs and enables the formation of female inflorescences. Altogether, this study presents a thorough overview of TCP family in maize and provides a new perspective on the evolution of this gene family. The results also indicate that TCP family genes may be involved in development stage in plant growing conditions. Additionally, our results will be useful for further functional analysis of the TCP gene family in maize.

Next-generation sequencing to solve complex inherited retinal dystrophy: A case series of multiple genes contributing to disease in extended families.

PubMed

Jones, Kaylie D; Wheaton, Dianna K; Bowne, Sara J; Sullivan, Lori S; Birch, David G; Chen, Rui; Daiger, Stephen P

2017-01-01

With recent availability of next-generation sequencing (NGS), it is becoming more common to pursue disease-targeted panel testing rather than traditional sequential gene-by-gene dideoxy sequencing. In this report, we describe using NGS to identify multiple disease-causing mutations that contribute concurrently or independently to retinal dystrophy in three relatively small families. Family members underwent comprehensive visual function evaluations, and genetic counseling including a detailed family history. A preliminary genetic inheritance pattern was assigned and updated as additional family members were tested. Family 1 (FAM1) and Family 2 (FAM2) were clinically diagnosed with retinitis pigmentosa (RP) and had a suspected autosomal dominant pedigree with non-penetrance (n.p.). Family 3 (FAM3) consisted of a large family with a diagnosis of RP and an overall dominant pedigree, but the proband had phenotypically cone-rod dystrophy. Initial genetic analysis was performed on one family member with traditional Sanger single gene sequencing and/or panel-based testing, and ultimately, retinal gene-targeted NGS was required to identify the underlying cause of disease for individuals within the three families. Results obtained in these families necessitated further genetic and clinical testing of additional family members to determine the complex genetic and phenotypic etiology of each family. Genetic testing of FAM1 (n = 4 affected; 1 n.p.) identified a dominant mutation in RP1 (p.Arg677Ter) that was present for two of the four affected individuals but absent in the proband and the presumed non-penetrant individual. Retinal gene-targeted NGS in the fourth affected family member revealed compound heterozygous mutations in USH2A (p. Cys419Phe, p.Glu767Serfs*21). Genetic testing of FAM2 (n = 3 affected; 1 n.p.) identified three retinal dystrophy genes ( PRPH2 , PRPF8 , and USH2A ) with disease-causing mutations in varying combinations among the affected family members. Genetic testing of FAM3 (n = 7 affected) identified a mutation in PRPH2 (p.Pro216Leu) tracking with disease in six of the seven affected individuals. Additional retinal gene-targeted NGS testing determined that the proband also harbored a multiple exon deletion in the CRX gene likely accounting for her cone-rod phenotype; her son harbored only the mutation in CRX , not the familial mutation in PRPH2 . Multiple genes contributing to the retinal dystrophy genotypes within a family were discovered using retinal gene-targeted NGS. Families with noted examples of phenotypic variation or apparent non-penetrant individuals may offer a clue to suspect complex inheritance. Furthermore, this finding underscores that caution should be taken when attributing a single gene disease-causing mutation (or inheritance pattern) to a family as a whole. Identification of a disease-causing mutation in a proband, even with a clear inheritance pattern in hand, may not be sufficient for targeted, known mutation analysis in other family members.

Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action

NASA Technical Reports Server (NTRS)

Yang, T.; Poovaiah, B. W.

2000-01-01

The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain. A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction.

GeneFarm, structural and functional annotation of Arabidopsis gene and protein families by a network of experts

PubMed Central

Aubourg, Sébastien; Brunaud, Véronique; Bruyère, Clémence; Cock, Mark; Cooke, Richard; Cottet, Annick; Couloux, Arnaud; Déhais, Patrice; Deléage, Gilbert; Duclert, Aymeric; Echeverria, Manuel; Eschbach, Aimée; Falconet, Denis; Filippi, Ghislain; Gaspin, Christine; Geourjon, Christophe; Grienenberger, Jean-Michel; Houlné, Guy; Jamet, Elisabeth; Lechauve, Frédéric; Leleu, Olivier; Leroy, Philippe; Mache, Régis; Meyer, Christian; Nedjari, Hafed; Negrutiu, Ioan; Orsini, Valérie; Peyretaillade, Eric; Pommier, Cyril; Raes, Jeroen; Risler, Jean-Loup; Rivière, Stéphane; Rombauts, Stéphane; Rouzé, Pierre; Schneider, Michel; Schwob, Philippe; Small, Ian; Soumayet-Kampetenga, Ghislain; Stankovski, Darko; Toffano, Claire; Tognolli, Michael; Caboche, Michel; Lecharny, Alain

2005-01-01

Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot. PMID:15608279

Conservation, Divergence, and Genome-Wide Distribution of PAL and POX A Gene Families in Plants.

PubMed

Rawal, H C; Singh, N K; Sharma, T R

2013-01-01

Genome-wide identification and phylogenetic and syntenic comparison were performed for the genes responsible for phenylalanine ammonia lyase (PAL) and peroxidase A (POX A) enzymes in nine plant species representing very diverse groups like legumes (Glycine max and Medicago truncatula), fruits (Vitis vinifera), cereals (Sorghum bicolor, Zea mays, and Oryza sativa), trees (Populus trichocarpa), and model dicot (Arabidopsis thaliana) and monocot (Brachypodium distachyon) species. A total of 87 and 1045 genes in PAL and POX A gene families, respectively, have been identified in these species. The phylogenetic and syntenic comparison along with motif distributions shows a high degree of conservation of PAL genes, suggesting that these genes may predate monocot/eudicot divergence. The POX A family genes, present in clusters at the subtelomeric regions of chromosomes, might be evolving and expanding with higher rate than the PAL gene family. Our analysis showed that during the expansion of POX A gene family, many groups and subgroups have evolved, resulting in a high level of functional divergence among monocots and dicots. These results will act as a first step toward the understanding of monocot/eudicot evolution and functional characterization of these gene families in the future.

Conservation, Divergence, and Genome-Wide Distribution of PAL and POX A Gene Families in Plants

PubMed Central

Rawal, H. C.; Singh, N. K.; Sharma, T. R.

2013-01-01

Genome-wide identification and phylogenetic and syntenic comparison were performed for the genes responsible for phenylalanine ammonia lyase (PAL) and peroxidase A (POX A) enzymes in nine plant species representing very diverse groups like legumes (Glycine max and Medicago truncatula), fruits (Vitis vinifera), cereals (Sorghum bicolor, Zea mays, and Oryza sativa), trees (Populus trichocarpa), and model dicot (Arabidopsis thaliana) and monocot (Brachypodium distachyon) species. A total of 87 and 1045 genes in PAL and POX A gene families, respectively, have been identified in these species. The phylogenetic and syntenic comparison along with motif distributions shows a high degree of conservation of PAL genes, suggesting that these genes may predate monocot/eudicot divergence. The POX A family genes, present in clusters at the subtelomeric regions of chromosomes, might be evolving and expanding with higher rate than the PAL gene family. Our analysis showed that during the expansion of POX A gene family, many groups and subgroups have evolved, resulting in a high level of functional divergence among monocots and dicots. These results will act as a first step toward the understanding of monocot/eudicot evolution and functional characterization of these gene families in the future. PMID:23671845

Identification and expression profile analysis of the sucrose phosphate synthase gene family in Litchi chinensis Sonn.

PubMed Central

Wang, Dan; Zhao, Jietang; Hu, Bing; Li, Jiaqi; Qin, Yaqi; Chen, Linhuan; Qin, Yonghua

2018-01-01

Sucrose phosphate synthase (SPS, EC 2.4.1.14) is a key enzyme that regulates sucrose biosynthesis in plants. SPS is encoded by different gene families which display differential expression patterns and functional divergence. Genome-wide identification and expression analyses of SPS gene families have been performed in Arabidopsis, rice, and sugarcane, but a comprehensive analysis of the SPS gene family in Litchi chinensis Sonn. has not yet been reported. In the current study, four SPS gene (LcSPS1, LcSPS2, LcSPS3, and LcSPS4) were isolated from litchi. The genomic organization analysis indicated the four litchi SPS genes have very similar exon-intron structures. Phylogenetic tree showed LcSPS1-4 were grouped into different SPS families (LcSPS1 and LcSPS2 in A family, LcSPS3 in B family, and LcSPS4 in C family). LcSPS1 and LcSPS4 were strongly expressed in the flowers, while LcSPS3 most expressed in mature leaves. RT-qPCR results showed that LcSPS genes expressed differentially during aril development between cultivars with different hexose/sucrose ratios. A higher level of expression of LcSPS genes was detected in Wuheli, which accumulates higher sucrose in the aril at mature. The tissue- and developmental stage-specific expression of LcSPS1-4 genes uncovered in this study increase our understanding of the important roles played by these genes in litchi fruits. PMID:29473005

Human cytochromes P450 in health and disease

PubMed Central

Nebert, Daniel W.; Wikvall, Kjell; Miller, Walter L.

2013-01-01

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

[Analysis of TGFBI gene mutation in a Chinese family affected with Reis-Bucklers corneal dystrophy].

PubMed

Guan, Tao; Zhang, Lingjie; Xu, Dejian; Wu, Haijian; Zheng, Libin

2017-10-10

To analyze the clinical features and TGFBI gene mutation in a Chinese family affected with Reis-Bucklers corneal dystrophy. Genomic DNA was extracted from 53 members including 9 patients from the family. The 17 exons and splice region of introns of the TGFBI gene were amplified by PCR and directly sequenced. All family members were subjected to ophthalmologic examination. A heterozygous mutation (R124L) was found in exon 4 of the TGFBI gene among all patients from the family. The same mutation was not found among unaffected family members. The inheritance pattern of the family was identified as autosomal dominant, and the Reis-Bucklers corneal dystrophy in the family was diagnosed as the geographic type. The R124L mutation of the TGFBI gene probably underlies the pathogenesis of Reis-Bucklers corneal dystrophy in this Chinese family. Molecular genetic approach is useful for the proper diagnosis of this type of corneal dystrophy.

Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms[OPEN

PubMed Central

Li, Zhen; Van de Peer, Yves; De Smet, Riet

2016-01-01

Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of “gene duplicability” is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. PMID:26744215

Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms.

PubMed

Li, Zhen; Defoort, Jonas; Tasdighian, Setareh; Maere, Steven; Van de Peer, Yves; De Smet, Riet

2016-02-01

Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of "gene duplicability" is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. © 2016 American Society of Plant Biologists. All rights reserved.

Relationships between IgE/IgG4 Epitopes, Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

PubMed Central

García-Mayoral, María Flor; Treviño, Miguel Angel; Pérez-Piñar, Teresa; Caballero, María Luisa; Knaute, Tobias; Umpierrez, Ana

2014-01-01

Background Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions. Methodology/Principal Findings The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera. Conclusions/Significance This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies. PMID:24603892

Structural, functional and evolutionary characterization of major drought transcription factors families in maize

NASA Astrophysics Data System (ADS)

Mittal, Shikha; Banduni, Pooja; Mallikarjuna, Mallana G.; Rao, Atmakuri R.; Jain, Prashant A.; Dash, Prasanta K.; Thirunavukkarasu, Nepolean

2018-05-01

Drought is one of the major threats to maize production. In order to improve the production and to breed tolerant hybrids, understanding the genes and regulatory mechanisms during drought stress is important. Transcription factors (TFs) play a major role in gene regulation and many TFs have been identified in response to drought stress. In our experiment, a set of 15 major TF families comprising 1436 genes was structurally and functionally characterized using in-silico tools and a gene expression assay. All 1436 genes were mapped on 10 chromosome of maize. The functional annotation indicated the involvement of these genes in ABA signaling, ROS scavenging, photosynthesis, stomatal regulation, and sucrose metabolism. Duplication was identified as the primary force in divergence and expansion of TF families. Phylogenetic relationship was developed individually for each TF family as well as combined TF families. Phylogenetic analysis grouped the TF family of genes into TF-specific and mixed groups. Phylogenetic analysis of genes belonging to various TF families suggested that the origin of TFs occurred in the lineage of maize evolution. Gene structure analysis revealed that more number of genes were intron-rich as compared to intronless genes. Drought-responsive CRE’s such as ABREA, ABREB, DRE1 and DRECRTCOREAT have been identified. Expression and interaction analyses identified leaf-specific bZIP TF, GRMZM2G140355, as a potential contributor toward drought tolerance in maize. We also analyzed protein-protein interaction network of 269 drought-responsive genes belonging to different drought-related TFs. The information generated on structural and functional characteristics, expression and interaction of the drought-related TF families will be useful to decipher the drought tolerance mechanisms and to derive drought-tolerant genotypes in maize.

Saltatory Evolution of the Ectodermal Neural Cortex Gene Family at the Vertebrate Origin

PubMed Central

Feiner, Nathalie; Murakami, Yasunori; Breithut, Lisa; Mazan, Sylvie; Meyer, Axel; Kuraku, Shigehiro

2013-01-01

The ectodermal neural cortex (ENC) gene family, whose members are implicated in neurogenesis, is part of the kelch repeat superfamily. To date, ENC genes have been identified only in osteichthyans, although other kelch repeat-containing genes are prevalent throughout bilaterians. The lack of elaborate molecular phylogenetic analysis with exhaustive taxon sampling has obscured the possible link of the establishment of this gene family with vertebrate novelties. In this study, we identified ENC homologs in diverse vertebrates by means of database mining and polymerase chain reaction screens. Our analysis revealed that the ENC3 ortholog was lost in the basal eutherian lineage through single-gene deletion and that the triplication between ENC1, -2, and -3 occurred early in vertebrate evolution. Including our original data on the catshark and the zebrafish, our comparison revealed high conservation of the pleiotropic expression pattern of ENC1 and shuffling of expression domains between ENC1, -2, and -3. Compared with many other gene families including developmental key regulators, the ENC gene family is unique in that conventional molecular phylogenetic inference could identify no obvious invertebrate ortholog. This suggests a composite nature of the vertebrate-specific gene repertoire, consisting not only of de novo genes introduced at the vertebrate origin but also of long-standing genes with no apparent invertebrate orthologs. Some of the latter, including the ENC gene family, may be too rapidly evolving to provide sufficient phylogenetic signals marking orthology to their invertebrate counterparts. Such gene families that experienced saltatory evolution likely remain to be explored and might also have contributed to phenotypic evolution of vertebrates. PMID:23843192

Basic Helix-Loop-Helix Transcription Factor Gene Family Phylogenetics and Nomenclature

PubMed Central

Skinner, Michael K.; Rawls, Alan; Wilson-Rawls, Jeanne; Roalson, Eric H.

2010-01-01

A phylogenetic analysis of the basic helix-loop-helix (bHLH) gene superfamily was performed using seven different species (human, mouse, rat, worm, fly, yeast, and plant Arabidopsis) and involving over 600 bHLH genes [1]. All bHLH genes were identified in the genomes of the various species, including expressed sequence tags, and the entire coding sequence was used in the analysis. Nearly 15% of the gene family has been updated or added since the original publication. A super-tree involving six clades and all structural relationships was established and is now presented for four of the species. The wealth of functional data available for members of the bHLH gene superfamily provides us with the opportunity to use this exhaustive phylogenetic tree to predict potential functions of uncharacterized members of the family. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique elements of the evolution and functional relationships of the different genes in the bHLH gene family. PMID:20219281

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

PubMed

Asquith, Christopher R M; Godoi, Paulo H; Couñago, Rafael M; Laitinen, Tuomo; Scott, John W; Langendorf, Christopher G; Oakhill, Jonathan S; Drewry, David H; Zuercher, William J; Koutentis, Panayiotis A; Willson, Timothy M; Kalogirou, Andreas S

2018-05-19

We demonstrate for the first time that 4 H -1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4 H -1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Genomic predictors of the maximal O2 uptake response to standardized exercise training programs

PubMed Central

Sarzynski, Mark A.; Rice, Treva K.; Kraus, William E.; Church, Timothy S.; Sung, Yun Ju; Rao, D. C.; Rankinen, Tuomo

2011-01-01

Low cardiorespiratory fitness is a powerful predictor of morbidity and cardiovascular mortality. In 473 sedentary adults, all whites, from 99 families of the Health, Risk Factors, Exercise Training, and Genetics (HERITAGE) Family Study, the heritability of gains in maximal O2 uptake (V̇o2max) after exposure to a standardized 20-wk exercise program was estimated at 47%. A genome-wide association study based on 324,611 single-nucleotide polymorphisms (SNPs) was undertaken to identify SNPs associated with improvements in V̇o2max Based on single-SNP analysis, 39 SNPs were associated with the gains with P < 1.5 × 10−4. Stepwise multiple regression analysis of the 39 SNPs identified a panel of 21 SNPs that accounted for 49% of the variance in V̇o2max trainability. Subjects who carried ≤9 favorable alleles at these 21 SNPs improved their V̇o2max by 221 ml/min, whereas those who carried ≥19 of these alleles gained, on average, 604 ml/min. The strongest association was with rs6552828, located in the acyl-CoA synthase long-chain member 1 (ACSL1) gene, which accounted by itself for ∼6% of the training response of V̇o2max. The genes nearest to the SNPs that were the strongest predictors were PR domain-containing 1 with ZNF domain (PRDM1); glutamate receptor, ionotropic, N-methyl-d-aspartate 3A (GRIN3A); K+ channel, voltage gated, subfamily H, member 8 (KCNH8); and zinc finger protein of the cerebellum 4 (ZIC4). The association with the SNP nearest to ZIC4 was replicated in 40- to 65-yr-old, sedentary, overweight, and dyslipidemic subjects trained in Studies of a Targeted Risk Reduction Intervention Through Defined Exercise (STRRIDE; n = 183). Two SNPs were replicated in sedentary obese white women exercise trained in the Dose Response to Exercise (DREW) study (n = 112): rs1956197 near dishevelled associated activator of morphogenesis 1 (DAAM1) and rs17117533 in the vicinity of necdin (NDN). The association of SNPs rs884736 in the calmodulin-binding transcription activator 1 (CAMTA1) locus and rs17581162 ∼68 kb upstream from regulator of G protein signaling 18 (RGS18) with the gains in V̇o2max in HERITAGE whites were replicated in HERITAGE blacks (n = 247). These genomic predictors of the response of V̇o2max to regular exercise provide new targets for the study of the biology of fitness and its adaptation to regular exercise. Large-scale replication studies are warranted. PMID:21183627

The Rice B-Box Zinc Finger Gene Family: Genomic Identification, Characterization, Expression Profiling and Diurnal Analysis

PubMed Central

Huang, Jianyan; Zhao, Xiaobo; Weng, Xiaoyu; Wang, Lei; Xie, Weibo

2012-01-01

Background The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now. Methodology/Principal Findings In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern. Conclusions/Significance The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes. PMID:23118960

Regulation of gene expression by dietary Ca2+ in kidneys of 25-hydroxyvitamin D3-1 alpha-hydroxylase knockout mice.

PubMed

Hoenderop, Joost G J; Chon, Helena; Gkika, Dimitra; Bluyssen, Hans A R; Holstege, Frank C P; St-Arnaud, Rene; Braam, Branko; Bindels, Rene J M

2004-02-01

Pseudovitamin D deficiency rickets (PDDR) is an autosomal disease, characterized by undetectable levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), rickets and secondary hyperparathyroidism. Mice in which the 25-hydroxyvitamin D3-1 alpha-hydroxylase (1 alpha-OHase) gene was inactivated, presented the same clinical phenotype as patients with PDDR. cDNA Microarray technology was used on kidneys of 1 alpha-OHase knockout mice to study the expression profile of renal genes in this Ca2+-related disorder. Genome wide molecular events that occur during the rescue of these mice by high dietary Ca2+ intake were studied by the use of 15K cDNA microarray chips. 1 alpha-OHase knockout mice fed a normal Ca2+ diet developed severe hypocalcemia, rickets and died with an average life span of 12 +/- 2 weeks. Intriguingly, 1 alpha-OHase-/- mice supplemented with an enriched Ca2+ diet were normocalcemic and not significantly different from wild-type mice. Inactivation of the 1 alpha-OHase gene resulted in a significant regulation of +/- 1000 genes, whereas dietary Ca2+ supplementation of the 1 alpha-OHase-/- mice revealed +/- 2000 controlled genes. Interestingly, 557 transcripts were regulated in both situations implicating the involvement in the dietary Ca2+-mediated rescue mechanism of the 1 alpha-OHase-/- mice. Conspicuous regulated genes encoded for signaling molecules like the PDZ-domain containing protein channel interacting protein, FK binding protein type 4, kinases, and importantly Ca2+ transporting proteins including the Na+-Ca2+ exchanger, calbindin-D28K and the Ca2+ sensor calmodulin. Dietary Ca2+ intake normalized disturbances in the Ca2+ homeostasis due to vitamin D deficiency that were accompanied by the regulation of a subset of renal genes, including well-known renal Ca2+ transport protein genes, but also genes not previously identified as playing a role in renal Ca2+ handling.

The Genetic Causes of Nonsyndromic Congenital Retinal Detachment: A Genetic and Phenotypic Study of Pakistani Families.

PubMed

Keser, Vafa; Khan, Ayesha; Siddiqui, Sorath; Lopez, Irma; Ren, Huanan; Qamar, Raheel; Nadaf, Javad; Majewski, Jacek; Chen, Rui; Koenekoop, Robert K

2017-02-01

To evaluate consanguineous pedigrees from Pakistan with a clinical diagnosis of nonsyndromic congenital retinal nonattachment (NCRNA) and identify genes responsible for the disease as currently only one NCRNA gene is known (atonal basic helix-loop-helix transcription factor 7: ATOH7). We implemented a three-step genotyping platform: single nucleotide polymorphism genotyping to identify loss of heterozygosity regions in patients, Retinal Information Network panel screening for mutations in currently known retinal genes. Negative patients were then subjected to whole exome sequencing. We evaluated 21 consanguineous NCRNA pedigrees and identified the causal mutations in known retinal genes in 13 out of our 21 families. We found mutations in ATOH7 in three families. Surprisingly, we then found mutations in familial exudative vitreoretinopathy (FEVR) genes; low-density lipoprotein receptor-related protein 5 mutations (six families), tetraspanin 12 mutations (two families), and NDP mutations (two families). Thus, 62% of the patients were successfully genotyped in our study with seven novel and six previously reported mutations in known retinal genes. Although the clinical diagnosis of all children was NCRNA with severe congenital fibrotic retinal detachments, the molecular diagnosis determined that the disease process was in fact a very severe form of FEVR in 10 families. Because severe congenital retinal detachment has not been previously associated with all the FEVR genes, we have thus expanded the phenotypic spectrum of FEVR, a highly variable retinal detachment phenotype that has clinical overlap with NCRNA. We identified seven novel mutations. We also established for the first time genetic overlap between the Iranian and Pakistani populations. We identified eight NCRNA families that do not harbor mutations in any known retinal genes, suggesting novel causal genes in these families.

The Genetic Causes of Nonsyndromic Congenital Retinal Detachment: A Genetic and Phenotypic Study of Pakistani Families

PubMed Central

Keser, Vafa; Khan, Ayesha; Siddiqui, Sorath; Lopez, Irma; Ren, Huanan; Qamar, Raheel; Nadaf, Javad; Majewski, Jacek; Chen, Rui; Koenekoop, Robert K.

2017-01-01

Purpose To evaluate consanguineous pedigrees from Pakistan with a clinical diagnosis of nonsyndromic congenital retinal nonattachment (NCRNA) and identify genes responsible for the disease as currently only one NCRNA gene is known (atonal basic helix-loop-helix transcription factor 7: ATOH7). Methods We implemented a three-step genotyping platform: single nucleotide polymorphism genotyping to identify loss of heterozygosity regions in patients, Retinal Information Network panel screening for mutations in currently known retinal genes. Negative patients were then subjected to whole exome sequencing. Results We evaluated 21 consanguineous NCRNA pedigrees and identified the causal mutations in known retinal genes in 13 out of our 21 families. We found mutations in ATOH7 in three families. Surprisingly, we then found mutations in familial exudative vitreoretinopathy (FEVR) genes; low-density lipoprotein receptor-related protein 5 mutations (six families), tetraspanin 12 mutations (two families), and NDP mutations (two families). Thus, 62% of the patients were successfully genotyped in our study with seven novel and six previously reported mutations in known retinal genes. Conclusions Although the clinical diagnosis of all children was NCRNA with severe congenital fibrotic retinal detachments, the molecular diagnosis determined that the disease process was in fact a very severe form of FEVR in 10 families. Because severe congenital retinal detachment has not been previously associated with all the FEVR genes, we have thus expanded the phenotypic spectrum of FEVR, a highly variable retinal detachment phenotype that has clinical overlap with NCRNA. We identified seven novel mutations. We also established for the first time genetic overlap between the Iranian and Pakistani populations. We identified eight NCRNA families that do not harbor mutations in any known retinal genes, suggesting novel causal genes in these families. PMID:28192794

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families

PubMed Central

Kahrizi, Kimia; Musante, Luciana; Fattahi, Zohreh; Hosseini, Masoumeh; Maqsoud, Fariba; Farajollahi, Reza; Wienker, Thomas F.; Ropers, H. Hilger; Najmabadi, Hossein

2015-01-01

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1–3% of the world’s population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder. PMID:26308914

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families.

PubMed

Davarniya, Behzad; Hu, Hao; Kahrizi, Kimia; Musante, Luciana; Fattahi, Zohreh; Hosseini, Masoumeh; Maqsoud, Fariba; Farajollahi, Reza; Wienker, Thomas F; Ropers, H Hilger; Najmabadi, Hossein

2015-01-01

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1-3% of the world's population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.

Genome-wide analysis of soybean HD-Zip gene family and expression profiling under salinity and drought treatments.

PubMed

Chen, Xue; Chen, Zhu; Zhao, Hualin; Zhao, Yang; Cheng, Beijiu; Xiang, Yan

2014-01-01

Homeodomain-leucine zipper (HD-Zip) proteins, a group of homeobox transcription factors, participate in various aspects of normal plant growth and developmental processes as well as environmental responses. To date, no overall analysis or expression profiling of the HD-Zip gene family in soybean (Glycine max) has been reported. An investigation of the soybean genome revealed 88 putative HD-Zip genes. These genes were classified into four subfamilies, I to IV, based on phylogenetic analysis. In each subfamily, the constituent parts of gene structure and motif were relatively conserved. A total of 87 out of 88 genes were distributed unequally on 20 chromosomes with 36 segmental duplication events, indicating that segmental duplication is important for the expansion of the HD-Zip family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the HD-Zip family basically underwent purifying selection with restrictive functional divergence after the duplication events. Analysis of expression profiles showed that 80 genes differentially expressed across 14 tissues, and 59 HD-Zip genes are differentially expressed under salinity and drought stress, with 20 paralogous pairs showing nearly identical expression patterns and three paralogous pairs diversifying significantly under drought stress. Quantitative real-time RT-PCR (qRT-PCR) analysis of six paralogous pairs of 12 selected soybean HD-Zip genes under both drought and salinity stress confirmed their stress-inducible expression patterns. This study presents a thorough overview of the soybean HD-Zip gene family and provides a new perspective on the evolution of this gene family. The results indicate that HD-Zip family genes may be involved in many plant responses to stress conditions. Additionally, this study provides a solid foundation for uncovering the biological roles of HD-Zip genes in soybean growth and development.

Identification and analysis of the TIFY gene family in Gossypium raimondii.

PubMed

He, D H; Lei, Z P; Tang, B S; Xing, H Y; Zhao, J X; Jing, Y L

2015-08-21

The highly conserved TIFY domain is included in the TIFY protein family of transcription factors, which is important in plant development. Here, 28 TIFY family genes were identified in the Gossypium raimondii genome and classified into JAZ (15 genes), ZML (8), PPD (3), and TIFY (2). The normal (TIF[F/Y]XG) motif was dominant in the TIFY family, excluding the ZML subfamily, in which TLSFXG was prevalent. TIFY family genes were unevenly distributed in the G. raimondii genome, with TIFY clusters present on chromosome 9. Phylogenetic analysis indicated abundant variations in the G. raimondii TIFY family, which were most closely related to those in Theobroma cacao among 5 species. Exon-intron organization and intron phases were homologous within each subfamily, correlating with their phylogeny. Intra-species synteny analyses indicated that genomic duplication contributed to the expansion of the TIFY family. Inter-species synteny analyses indicated that synteny regions involved in G. raimondii TIFY family genes were also present in the comparison of G. raimondii vs Arabidopsis thaliana or T. cacao, signifying that these genes had common ancestors and play the same or similar roles in biological processes. Greater synteny was present in the comparison of G. raimondii vs T. cacao than of G. raimondii vs A. thaliana. The expression patterns of TIFY family genes were characterized and most TIFY family genes were indicated to be involved in fiber development. Our study provides new data related to the evolution of TIFYs and their role as important regulators of transcription; these data can be useful for fiber development.

Neurobehavioral changes and alteration of gene expression in the brains of metallothionein-I/II null mice exposed to low levels of mercury vapor during postnatal development.

PubMed

Yoshida, Minoru; Honda, Masako; Watanabe, Chiho; Satoh, Masahiko; Yasutake, Akira

2011-10-01

This study examined the neurobehavioral changes and alteration in gene expression in the brains of metallothionein (MT)-I/II null mice exposed to low-levels of mercury vapor (Hg(0)) during postnatal development. MT-I/II null and wild-type mice were repeatedly exposed to Hg(0) at 0.030 mg/m(3) (range: 0.023-0.043 mg/m(3)), which was similar to the current threshold value (TLV), for 6 hr per day until the 20th day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. Hg(0)-exposed MT-I/II null mice showed a significant decrease in total locomotor activity in females, though learning ability and spatial learning ability were not affected. Immediately after Hg(0) exposure, mercury concentrations in the brain did not exceed 0.5 µg/g in any animals. Hg(0) exposure resulted in significant alterations in gene expression in the brains of both strains using DNA microarray analysis. The number of altered genes in MT-I/II null mice was higher than that in wild-type mice and calcium-calmodulin kinase II (Camk2a) involved in learning and memory in down-regulated genes was detected. These results provide useful information to elucidate the development of behavioral toxicity following low-level exposure to Hg(0).

Genome-wide identification and expression profiling of dehydrin gene family in Malus domestica.

PubMed

Liang, Dong; Xia, Hui; Wu, Shan; Ma, Fengwang

2012-12-01

The family of dehydrin genes has important roles in protecting higher plants against abiotic stress, such as drought, salinity and cold. However, knowledge about apple dehydrin gene family is limited. In the present study, we used a bioinformatics approach to identify members of that family in apple (Malus domestica). A total of 12 apple dehydrin genes (MdDHNs) were identified and located on various chromosomes. All putative proteins from those genes contained a typical K domain. Among 12 MdDHNs, nine were cloned and their expression patterns were investigated. Expression profiling indicated that the these nine dehydrin genes display differential expression patterns in various tissues. Moreover, transcript levels of some MdDHNs were up-regulated significantly under drought, low temperature, or ABA treatment, which indicated their important roles during stress adaptation. These results demonstrate that the apple dehydrin gene family may function in tissue development and plant stress responses.

Calcium Input Frequency, Duration and Amplitude Differentially Modulate the Relative Activation of Calcineurin and CaMKII

PubMed Central

Li, Lu; Stefan, Melanie I.; Le Novère, Nicolas

2012-01-01

NMDA receptor dependent long-term potentiation (LTP) and long-term depression (LTD) are two prominent forms of synaptic plasticity, both of which are triggered by post-synaptic calcium elevation. To understand how calcium selectively stimulates two opposing processes, we developed a detailed computational model and performed simulations with different calcium input frequencies, amplitudes, and durations. We show that with a total amount of calcium ions kept constant, high frequencies of calcium pulses stimulate calmodulin more efficiently. Calcium input activates both calcineurin and Ca2+/calmodulin-dependent protein kinase II (CaMKII) at all frequencies, but increased frequencies shift the relative activation from calcineurin to CaMKII. Irrespective of amplitude and duration of the inputs, the total amount of calcium ions injected adjusts the sensitivity of the system to calcium input frequencies. At a given frequency, the quantity of CaMKII activated is proportional to the total amount of calcium. Thus, an input of a small amount of calcium at high frequencies can induce the same activation of CaMKII as a larger amount, at lower frequencies. Finally, the extent of activation of CaMKII signals with high calcium frequency is further controlled by other factors, including the availability of calmodulin, and by the potency of phosphatase inhibitors. PMID:22962589

Development of the fluorescent biosensor hCalmodulin (hCaM)L39C-monobromobimane(mBBr)/V91C-mBBr, a novel tool for discovering new calmodulin inhibitors and detecting calcium.

PubMed

Gonzalez-Andrade, Martin; Rivera-Chavez, Jose; Sosa-Peinado, Alejandro; Figueroa, Mario; Rodriguez-Sotres, Rogelio; Mata, Rachel

2011-06-09

A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca(2+)) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca(2+) on trifluoroperazine (TFP) binding to CaM (Ca(2+)K(d) = 0.24 μM ± 0.03 with a stoichiometry 4.10 ± 0.15; TFPK(d) ∼ 5.74-0.53 μM depending on the degree of saturation of Ca(2+), with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 1-5, 7, and 8 for the complex (Ca(2+))(4)-CaM was excellent because their experimental K(d)s were in the nM range (4-498 nM). Docking analysis predicted that 1-8 bind to CaM at sites I, III, and IV as does TFP.

Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor

PubMed Central

Hashimoto, Y; Nawa, M; Kurita, M; Tokizawa, M; Iwamatsu, A; Matsuoka, M

2013-01-01

Humanin is a secreted bioactive peptide that is protective in a variety of death models, including cell-based neuronal death models related to Alzheimer's disease (AD). To mediate the protective effect in AD-related death models, Humanin signals via a cell-surface receptor that is generally composed of three subunits: ciliary neurotrophic factor receptor α, WSX-1 and gp130 (heterotrimeric Humanin receptor; htHNR). However, the protective effect of Humanin via the htHNR is weak (EC50=1–10 μℳ); therefore, it is possible that another physiological agonist for this receptor exists in vivo. In the current study, calmodulin-like skin protein (CLSP), a calmodulin relative with an undefined function, was shown to be secreted and inhibit neuronal death via the htHNR with an EC50 of 10–100 pℳ. CLSP was highly expressed in the skin, and the concentration in circulating normal human blood was ∼5 nℳ. When administered intraperitoneally in mice, recombinant CLSP was transported across the blood-cerebrospinal fluid (CSF)-barrier and its concentration in the CSF reaches 1/100 of its serum concentration at 1 h after injection. These findings suggest that CLSP is a physiological htHNR agonist. PMID:23519124

Cotranslocational processing of the protein substrate calmodulin by an AAA+ unfoldase occurs via unfolding and refolding intermediates.

PubMed

Augustyniak, Rafal; Kay, Lewis E

2018-05-22

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments.

A Postpartum Model in Rat: Behavioral and Gene Expression Changes Induced by Ovarian Steroid Deprivation

PubMed Central

Suda, Shiro; Segi-Nishida, Eri; Newton, Samuel S.; Duman, Ronald S.

2013-01-01

Background Postpartum depression (PPD) affects approximately 10% to 20% of women during the first 4 weeks of the postpartum period and is characterized by labile mood with prominent anxiety and irritability, insomnia,and depressive mood. During the postpartum period, elevated ovarian hormones abruptly decrease to the early follicular phase levels that are postulated to play a major role in triggering PPD. However, the underlying neurobiological mechanisms that contribute to PPD have not been determined. Methods In the present study, we examined the effect of ovarian steroids, administered at levels that occur during human pregnancy followed by rapid withdrawal to simulate postpartum conditions, on behavior and gene expression in the rat. Results The results of behavioral testing reveal that the hormone-simulated postpartum treatment results in the development of a phenotype relevant to PPD, including vulnerability for helplessness, increased anxiety, and aggression. Real-time quantitative polymerase chain reaction (PCR) demonstrated transient regulation of several genes, including Ca2+/calmodulin-dependent protein kinase II (CAMKII), serotonin transporter (SERT), myocyte enhancer factor 2A (MEF2A), brain-derived neurotrophic factor (BDNF), gamma-aminobutyric acid type A receptor α4 (GABAARA4), mothers against decapentaplegic homolog 4 (SMAD4), and aquaporin 4 (AQP4) that could underlie these behavioral effects. Conclusions These studies provide an improved understanding of the effects of withdrawal from high doses of ovarian hormones on behavior and gene expression changes in the brain that could contribute to the pathophysiology of PPD. PMID:18471802

Revisiting the diffusion approximation to estimate evolutionary rates of gene family diversification.

PubMed

Gjini, Erida; Haydon, Daniel T; David Barry, J; Cobbold, Christina A

2014-01-21

Genetic diversity in multigene families is shaped by multiple processes, including gene conversion and point mutation. Because multi-gene families are involved in crucial traits of organisms, quantifying the rates of their genetic diversification is important. With increasing availability of genomic data, there is a growing need for quantitative approaches that integrate the molecular evolution of gene families with their higher-scale function. In this study, we integrate a stochastic simulation framework with population genetics theory, namely the diffusion approximation, to investigate the dynamics of genetic diversification in a gene family. Duplicated genes can diverge and encode new functions as a result of point mutation, and become more similar through gene conversion. To model the evolution of pairwise identity in a multigene family, we first consider all conversion and mutation events in a discrete manner, keeping track of their details and times of occurrence; second we consider only the infinitesimal effect of these processes on pairwise identity accounting for random sampling of genes and positions. The purely stochastic approach is closer to biological reality and is based on many explicit parameters, such as conversion tract length and family size, but is more challenging analytically. The population genetics approach is an approximation accounting implicitly for point mutation and gene conversion, only in terms of per-site average probabilities. Comparison of these two approaches across a range of parameter combinations reveals that they are not entirely equivalent, but that for certain relevant regimes they do match. As an application of this modelling framework, we consider the distribution of nucleotide identity among VSG genes of African trypanosomes, representing the most prominent example of a multi-gene family mediating parasite antigenic variation and within-host immune evasion. © 2013 Published by Elsevier Ltd. All rights reserved.

Genome-wide identification, expansion, and evolution analysis of homeobox genes and their expression profiles during root development in carrot.

PubMed

Que, Feng; Wang, Guang-Long; Li, Tong; Wang, Ya-Hui; Xu, Zhi-Sheng; Xiong, Ai-Sheng

2018-06-16

The homeobox gene family, a large family represented by transcription factors, has been implicated in secondary growth, early embryo patterning, and hormone response pathways in plants. However, reports about the information and evolutionary history of the homeobox gene family in carrot are limited. In the present study, a total of 130 homeobox family genes were identified in the carrot genome. Specific codomain and phylogenetic analyses revealed that the genes were classified into 14 subgroups. Whole genome and proximal duplication participated in the homeobox gene family expansion in carrot. Purifying selection also contributed to the evolution of carrot homeobox genes. In Gene Ontology (GO) analysis, most members of the HD-ZIP III and IV subfamilies were found to have a lipid binding (GO:0008289) term. Most HD-ZIP III and IV genes also harbored a steroidogenic acute regulatory protein-related lipid transfer (START) domain. These results suggested that the HD-ZIP III and IV subfamilies might be related to lipid transfer. Transcriptome and quantitative real-time PCR (RT-qPCR) data indicated that members of the WOX and KNOX subfamilies were likely implicated in carrot root development. Our study provided a useful basis for further studies on the complexity and function of the homeobox gene family in carrot.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

PubMed Central

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle.

PubMed

Rezaei, Hossein B; Kamato, Danielle; Ansari, Ghazaleh; Osman, Narin; Little, Peter J

2012-08-01

The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

DMT efficiently inhibits hepatic gluconeogenesis by regulating the Gαq signaling pathway.

PubMed

Zhou, Ting-Ting; Ma, Fei; Shi, Xiao-Fan; Xu, Xin; Du, Te; Guo, Xiao-Dan; Wang, Gai-Hong; Yu, Liang; Rukachaisirikul, Vatcharin; Hu, Li-Hong; Chen, Jing; Shen, Xu

2017-08-01

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with complicated pathogenesis and targeting gluconeogenesis inhibition is a promising strategy for anti-diabetic drug discovery. G protein-coupled receptors (GPCRs) are classified as distinct families by heterotrimeric G proteins, primarily including Gαs, Gαi and Gαq. Gαs-coupled GPCRs function potently in the regulation of hepatic gluconeogenesis by activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and Gαi-coupled GPCRs exhibit inhibitory effect on adenylyl cyclase and reduce intracellular cAMP level. However, little is known about the regulation of Gαq-coupled GPCRs in hepatic gluconeogenesis. Here, small-molecule 2-(2,4-dimethoxy-3-methylphenyl)-7-(thiophen-2-yl)-9-(trifluoromethyl)-2,3-dihydropyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4( 1H )-one (DMT) was determined to suppress hepatic glucose production and reduce mRNA levels of gluconeogenic genes. Treatment of DMT in db/db mice decreased fasting blood glucose and hemoglobin A1C (HbA1c) levels, while improved glucose tolerance and pyruvate tolerance. Mechanism study demonstrated that DMT-inhibited gluconeogenesis by regulating the Gαq/phospholipase C (PLC)/inositol-1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca 2+ )/calmodulin (CaM)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box protein O1 (FOXO1) signaling pathway. To our knowledge, DMT might be the first reported small molecule able to suppress hepatic gluconeogenesis by regulating Gαq signaling, and our current work has also highlighted the potential of DMT in the treatment of T2DM. © 2017 Society for Endocrinology.

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

PubMed

Johnstone, Daniel; Graham, Ross M; Trinder, Debbie; Delima, Roheeth D; Riveros, Carlos; Olynyk, John K; Scott, Rodney J; Moscato, Pablo; Milward, Elizabeth A

2012-04-11

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Calpain 3 and CaMKIIβ signaling are required to induce HSP70 necessary for adaptive muscle growth after atrophy

PubMed Central

Kramerova, Irina; Torres, Jorge A; Eskin, Ascia; Nelson, Stanley F; Spencer, Melissa J

2018-01-01

Abstract Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIβ signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIβ signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIβ regulates remodeling in multiple contexts: endurance exercise and growth after atrophy. PMID:29528394

Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

PubMed

Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

2017-08-01

Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca 2+ productions, level of mitochondria membrane potential (ΔΨ m ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ m , and Ca 2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells. © 2016 Wiley Periodicals, Inc.

Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.).

PubMed

Mirza, Neelofar; Taj, Gohar; Arora, Sandeep; Kumar, Anil

2014-10-25

Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca(2+)/H(+) antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca(2+) ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca(2+) ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet. Copyright © 2014 Elsevier B.V. All rights reserved.

Genome-wide identification and analysis of the MADS-box gene family in apple.

PubMed

Tian, Yi; Dong, Qinglong; Ji, Zhirui; Chi, Fumei; Cong, Peihua; Zhou, Zongshan

2015-01-25

The MADS-box gene family is one of the most widely studied families in plants and has diverse developmental roles in flower pattern formation, gametophyte cell division and fruit differentiation. Although the genome-wide analysis of this family has been performed in some species, little is known regarding MADS-box genes in apple (Malus domestica). In this study, 146 MADS-box genes were identified in the apple genome and were phylogenetically clustered into six subgroups (MIKC(c), MIKC*, Mα, Mβ, Mγ and Mδ) with the MADS-box genes from Arabidopsis and rice. The predicted apple MADS-box genes were distributed across all 17 chromosomes at different densities. Additionally, the MADS-box domain, exon length, gene structure and motif compositions of the apple MADS-box genes were analysed. Moreover, the expression of all of the apple MADS-box genes was analysed in the root, stem, leaf, flower tissues and five stages of fruit development. All of the apple MADS-box genes, with the exception of some genes in each group, were expressed in at least one of the tissues tested, which indicates that the MADS-box genes are involved in various aspects of the physiological and developmental processes of the apple. To the best of our knowledge, this report describes the first genome-wide analysis of the apple MADS-box gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family. Copyright © 2014 Elsevier B.V. All rights reserved.

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

NASA Technical Reports Server (NTRS)

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.

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+•Calmodulin Displays a Unique Binding Mode*

PubMed Central

Liu, Yanshun; Zheng, Xunhai; Mueller, Geoffrey A.; Sobhany, Mack; DeRose, Eugene F.; Zhang, Yingpei; London, Robert E.; Birnbaumer, Lutz

2012-01-01

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2+-depletion sensor STIM1. This is followed by a fast Ca2+·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp76 of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1. PMID:23109337

Resveratrol Increases Nitric Oxide Production in the Rat Thick Ascending Limb via Ca2+/Calmodulin

PubMed Central

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 (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick 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. PMID:25314136

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

PubMed

Hentz, N G; Daunert, S

1996-11-15

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

SKF83959 Produces Antidepressant Effects in a Chronic Social Defeat Stress Model of Depression through BDNF-TrkB Pathway

PubMed Central

Jiang, Bo; Wang, Fang; Yang, Si; Fang, Peng; Deng, Zhi-Fang; Xiao, Jun-Li; Hu, Zhuang-Li

2015-01-01

Background: SKF83959 stimulates the phospholipase Cβ/inositol phosphate 3 pathway, resulting in the activation of Ca2+/calmodulin-dependent kinase IIα, which affects the synthesis of brain-derived neurotrophic factor, a neurotrophic factor critical for the pathophysiology of depression. Previous reports showed that SKF83959 elicited antidepressant activity in the forced swim test and tail suspension test as a novel triple reuptake inhibitor. However, there are no studies showing the effects of SKF83959 in a chronic stress model of depression and the role of phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway in SKF83959-mediated antidepressant effects. Methods: In this study, SKF83959 was firstly investigated in the chronic social defeat stress model of depression. The changes in hippocampal neurogenesis, dendrite spine density, and brain-derived neurotrophic factor signaling pathway after chronic social defeat stress and SKF83959 treatment were then investigated. Pharmacological inhibitors and small interfering RNA/short hairpin RNA methods were further used to explore the antidepressive mechanisms of SKF83959. Results: We found that SKF83959 produced antidepressant effects in the chronic social defeat stress model and also restored the chronic social defeat stress-induced decrease in hippocampal brain-derived neurotrophic factor signaling pathway, dendritic spine density, and neurogenesis. By using various inhibitors and siRNA/shRNA methods, we further demonstrated that the hippocampal dopamine D5 receptor, phospholipase C/inositol phosphate 3/ calmodulin-dependent kinase IIα pathway, and brain-derived neurotrophic factor system are all necessary for the SKF83959 effects. Conclusion: These results suggest that SKF83959 can be developed as a novel antidepressant and produces antidepressant effects via the hippocampal D5/ phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway. PMID:25522427

Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamb, A.; Weir, M.; Rudy, B.

1989-06-01

The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this methodmore » to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.« less

Lineage-Specific Evolutionary Histories and Regulation of Major Starch Metabolism Genes during Banana Ripening

PubMed Central

Jourda, Cyril; Cardi, Céline; Gibert, Olivier; Giraldo Toro, Andrès; Ricci, Julien; Mbéguié-A-Mbéguié, Didier; Yahiaoui, Nabila

2016-01-01

Starch is the most widespread and abundant storage carbohydrate in plants. It is also a major feature of cultivated bananas as it accumulates to large amounts during banana fruit development before almost complete conversion to soluble sugars during ripening. Little is known about the structure of major gene families involved in banana starch metabolism and their evolution compared to other species. To identify genes involved in banana starch metabolism and investigate their evolutionary history, we analyzed six gene families playing a crucial role in plant starch biosynthesis and degradation: the ADP-glucose pyrophosphorylases (AGPases), starch synthases (SS), starch branching enzymes (SBE), debranching enzymes (DBE), α-amylases (AMY) and β-amylases (BAM). Using comparative genomics and phylogenetic approaches, these genes were classified into families and sub-families and orthology relationships with functional genes in Eudicots and in grasses were identified. In addition to known ancestral duplications shaping starch metabolism gene families, independent evolution in banana and grasses also occurred through lineage-specific whole genome duplications for specific sub-families of AGPase, SS, SBE, and BAM genes; and through gene-scale duplications for AMY genes. In particular, banana lineage duplications yielded a set of AGPase, SBE and BAM genes that were highly or specifically expressed in banana fruits. Gene expression analysis highlighted a complex transcriptional reprogramming of starch metabolism genes during ripening of banana fruits. A differential regulation of expression between banana gene duplicates was identified for SBE and BAM genes, suggesting that part of starch metabolism regulation in the fruit evolved in the banana lineage. PMID:27994606

Molecular investigation of mental retardation locus gene PRSS12 by linkage analysis

PubMed Central

Ali, Zafar; Babar, Masroor Ellahi; Ahmad, Jamil; Yousaf, Muhammad Zubair; Asif, Muhammad; Shah, Sajjad Ali

2011-01-01

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked. PMID:22090715

Molecular investigation of mental retardation locus gene PRSS12 by linkage analysis.

PubMed

Ali, Zafar; Babar, Masroor Ellahi; Ahmad, Jamil; Yousaf, Muhammad Zubair; Asif, Muhammad; Shah, Sajjad Ali

2011-05-01

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked.

Streamlining and Large Ancestral Genomes in Archaea Inferred with a Phylogenetic Birth-and-Death Model

PubMed Central

Miklós, István

2009-01-01

Homologous genes originate from a common ancestor through vertical inheritance, duplication, or horizontal gene transfer. Entire homolog families spawned by a single ancestral gene can be identified across multiple genomes based on protein sequence similarity. The sequences, however, do not always reveal conclusively the history of large families. To study the evolution of complete gene repertoires, we propose here a mathematical framework that does not rely on resolved gene family histories. We show that so-called phylogenetic profiles, formed by family sizes across multiple genomes, are sufficient to infer principal evolutionary trends. The main novelty in our approach is an efficient algorithm to compute the likelihood of a phylogenetic profile in a model of birth-and-death processes acting on a phylogeny. We examine known gene families in 28 archaeal genomes using a probabilistic model that involves lineage- and family-specific components of gene acquisition, duplication, and loss. The model enables us to consider all possible histories when inferring statistics about archaeal evolution. According to our reconstruction, most lineages are characterized by a net loss of gene families. Major increases in gene repertoire have occurred only a few times. Our reconstruction underlines the importance of persistent streamlining processes in shaping genome composition in Archaea. It also suggests that early archaeal genomes were as complex as typical modern ones, and even show signs, in the case of the methanogenic ancestor, of an extremely large gene repertoire. PMID:19570746

Protective effect of ganodermanondiol isolated from the Lingzhi mushroom against tert-butyl hydroperoxide-induced hepatotoxicity through Nrf2-mediated antioxidant enzymes.

PubMed

Li, Bin; Lee, Dong-Sung; Kang, Yue; Yao, Nai-Qi; An, Ren-Bo; Kim, Youn-Chul

2013-03-01

Ganodermanondiol, a biologically active compound, was isolated from the Lingzhi mushroom (Ganoderma lucidum). The present study examined the protective effects of ganodermanondiol against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity. Ganodermanondiol protected human liver-derived HepG2 cells through nuclear factor-E2-related factor 2 (Nrf2) pathway-dependent heme oxygenase-1 expressions. Moreover, ganodermanondiol increased cellular glutathione levels and the expression of the glutamine-cysteine ligase gene in a dose-dependent manner. Furthermore, ganodermanondiol exposure enhanced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its upstream kinase activators, LKB1 and Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII). This study indicates that ganodermanondiol exhibits potent cytoprotective effects on t-BHP-induced hepatotoxicity in human liver-derived HepG2 cells, presumably through Nrf2-mediated antioxidant enzymes and AMPK. Copyright © 2012 Elsevier Ltd. All rights reserved.

Species of Phaeoacremonium Associated with Infections in Humans and Environmental Reservoirs in Infected Woody Plants

PubMed Central

Mostert, Lizel; Groenewald, Johannes Z.; Summerbell, Richard C.; Robert, Vincent; Sutton, Deanna A.; Padhye, Arvind A.; Crous, Pedro W.

2005-01-01

To date, three species of Phaeoacremonium have been associated with phaeohyphomycosis. These are P. parasiticum (formerly Phialophora parasitica), P. inflatipes, and P. rubrigenum. Numerous unknown isolates resembling Phaeoacremonium spp. have in recent years been isolated from human patients as well as from woody plants that appear to be the main environmental source of these fungi. Nine new Phaeoacremonium species, of which six were obtained as etiologic agents of human opportunistic infection, are reported. They can be identified based on their cultural and morphological characters, and the identifications are strongly supported in phylogenetic analyses of partial sequences of the actin, β-tubulin, and calmodulin genes. A multiple-entry electronic key based on morphological, cultural, and β-tubulin sequence data was developed to facilitate routine species identification. Reexamination of all isolates of P. inflatipes associated with human disease showed them to be misidentified and to belong to the new taxa described here. PMID:15814996

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

PubMed Central

Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F.; Lecuit, Marc

2016-01-01

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents. PMID:27177310

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.

PubMed

Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F; Lecuit, Marc

2016-05-12

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

Genetic variation analysis and relationships among environmental strains of Scedosporium apiospermum sensu stricto in Bangkok, Thailand.

PubMed

Wongsuk, Thanwa; Pumeesat, Potjaman; Luplertlop, Natthanej

2017-01-01

The Scedosporium apiospermum species complex is an emerging filamentous fungi that has been isolated from environment. It can cause a wide range of infections in both immunocompetent and immunocompromised individuals. We aimed to study the genetic variation and relationships between 48 strains of S. apiospermum sensu stricto isolated from soil in Bangkok, Thailand. For PCR, sequencing and phylogenetic analysis, we used the following genes: actin; calmodulin exons 3 and 4; the second largest subunit of the RNA polymerase II; ß-tubulin exon 2-4; manganese superoxide dismutase; internal transcribed spacer; transcription elongation factor 1α; and beta-tubulin exons 5 and 6. The present study is the first phylogenetic analysis of relationships among S. apiospermum sensu stricto in Thailand and South-east Asia. This result provides useful information for future epidemiological study and may be correlated to clinical manifestation.

Genetic variation analysis and relationships among environmental strains of Scedosporium apiospermum sensu stricto in Bangkok, Thailand

PubMed Central

2017-01-01

The Scedosporium apiospermum species complex is an emerging filamentous fungi that has been isolated from environment. It can cause a wide range of infections in both immunocompetent and immunocompromised individuals. We aimed to study the genetic variation and relationships between 48 strains of S. apiospermum sensu stricto isolated from soil in Bangkok, Thailand. For PCR, sequencing and phylogenetic analysis, we used the following genes: actin; calmodulin exons 3 and 4; the second largest subunit of the RNA polymerase II; ß-tubulin exon 2–4; manganese superoxide dismutase; internal transcribed spacer; transcription elongation factor 1α; and beta-tubulin exons 5 and 6. The present study is the first phylogenetic analysis of relationships among S. apiospermum sensu stricto in Thailand and South-east Asia. This result provides useful information for future epidemiological study and may be correlated to clinical manifestation. PMID:28704511

Mutants with Altered Sensitivity to a Calmodulin Antagonist Affect the Circadian Clock in Neurospora Crassa

PubMed Central

Suzuki, S.; Katagiri, S.; Nakashima, H.

1996-01-01

Two newly isolated mutant strains of Neurospora crassa, cpz-1 and cpz-2, were hypersensitive to chlorpromazine with respect to mycelial growth but responded differently to the drug with respect to the circadian conidiation rhythm. In the wild type, chlorpromazine caused shortening of the period length of the conidiation rhythm. Pulse treatment with the drug shifted the phase and inhibited light-induced phase shifting in Neurospora. By contrast to the wild type, the cpz-2 strain was resistant to these inhibitory effects of chlorpromazine. Inhibition of cpz-2 function by chlorpromazine affected three different parameters of circadian conidiation rhythm, namely, period length, phase and light-induced phase shifting. These results indicate that the cpz-2 gene must be involved in or related closely to the clock mechanism of Neurospora. By contrast, the cpz-1 strain was hypersensitive to chlorpromazine with respect to the circadian conidiation rhythm. PMID:8807291

The role of calcium and calmodulin in freezing-induced freezing resistance of Populus tomentosa cuttings.

PubMed

Lin, Shan-Zhi; Zhang, Zhi-Yi; Lin, Yuan-Zhen; Zhang, Qian; Guo, Huan

2004-02-01

To explore the role of calcium-calmodulin messenger system in the transduction of low temperature signal in woody plants, Populus tomentosa cuttings after being treated with CaCl(2) (10 mmol/L), Ca(2+) chelator EGTA (3 mmol/L), Ca(2+) channel inhibitor LaCl(3) (100 mmol/L) or CaM antagonist CPZ (50 mmol/L) were used for freezing acclimation at -3 degrees C. The changes in the calmodulin (CaM) and malonaldehyde (MDA) contents, the activities of superoxide dismutase (SOD), peroxidase (POD) and Ca(2+)-dependent adenosinetriphosphatase (Ca(2+)-ATPase) of mitochondrial membrane as well as freezing resistance (expressed as LT(50)) of cuttings were investigated to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation increased the CaM content, the activities of SOD, POD and Ca(2+)-ATPase of mitochondrial membrane as well as freezing resistance of cuttings, and decreased the MDA content as compared with control cuttings. Treatment with CaCl(2) at the time of freezing acclimation enhanced the effect of freezing acclimation on the above-mentioned indexes, but this enhancement was abolished by Ca(2+)chelator EGTA, Ca(2+) channel inhibitor LaCl(3) or CaM antagonist CPZ, indicating that the calcium-calmodulin messenger system was involved in the course of freezing resistance development. The presence of CaCl(2) at the same time of freezing acclimation also reduced the degree of decline in CaM content, and in SOD, POD and Ca(2+)-ATPase activities caused by freezing stress at -14 degrees C, and enhanced the level of increase in CaM content, and in SOD, POD and Ca(2+)-ATPase activity in the recovery periods at 25 degrees C . The change in CaM content was found to be closely correlated to the levels of SOD, POD and Ca(2+)-ATPase, and to the degree of freezing resistance of cuttings during freezing acclimation either with or without CaCl(2) treatment. It was suggested that the increase of CaM content induced by CaCl(2) treatment promote the formation of Ca(2+)-CaM complexes, which effectively activates the activities of SOD, POD and mitochondrial Ca(2+)-ATPase and then further result in the adaptive changes associated with the development and enhancement of freezing resistance. Thus, It could be concluded that Ca(2+)-calmodulin may be involved in the regulation of the increase in SOD, POD and Ca(2+)-ATPase activities, and the induction of freezing resistance of cuttings.

Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori)

PubMed Central

Tang, Xin; Liu, Huawei; Chen, Quanmei; Wang, Xin; Xiong, Ying; Zhao, Ping

2016-01-01

The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family. PMID:27706106

Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori).

PubMed

Tang, Xin; Liu, Huawei; Chen, Quanmei; Wang, Xin; Xiong, Ying; Zhao, Ping

2016-10-03

The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family.

Genetic diagnosis of high-penetrance susceptibility for colorectal cancer (CRC) is achievable for a high proportion of familial CRC by exome sequencing.

PubMed

Chubb, Daniel; Broderick, Peter; Frampton, Matthew; Kinnersley, Ben; Sherborne, Amy; Penegar, Steven; Lloyd, Amy; Ma, Yussanne P; Dobbins, Sara E; Houlston, Richard S

2015-02-10

Knowledge of the contribution of high-penetrance susceptibility to familial colorectal cancer (CRC) is relevant to the counseling, treatment, and surveillance of CRC patients and families. To quantify the impact of germline mutation to familial CRC, we sequenced the mismatch repair genes (MMR) APC, MUTYH, and SMAD4/BMPR1A in 626 early-onset familial CRC cases ascertained through a population-based United Kingdom national registry. In addition, we evaluated the contribution of mutations in the exonuclease domain (exodom) of POLE and POLD1 genes that have recently been reported to confer CRC risk. Overall mutations (pathogenic, likely pathogenic) in MMR genes make the highest contribution to familial CRC (10.9%). Mutations in the other established CRC genes account for 3.3% of cases. POLE/POLD1 exodom mutations were identified in three patients with family histories consistent with dominant transmission of CRC. Collectively, mutations in the known genes account for 14.2% of familial CRC (89 of 626 cases; 95% CI = 11.5, 17.2). A genetic diagnosis is feasible in a high proportion of familial CRC. Mainstreaming such analysis in clinical practice should enable the medical management of patients and their families to be optimized. Findings suggest CRC screening of POLE and POLD1 mutation carriers should be comparable to that afforded to those at risk of HNPCC. Although the risk of CRC associated with unexplained familial CRC is in general moderate, in some families the risk is substantive and likely to be the consequence of unidentified genes, as exemplified by POLE and POLD1. Our findings have utility in the design of genetic analyses to identify such novel CRC risk genes. © 2015 by American Society of Clinical Oncology.