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Thyroid hormones influence brain development through the regulation of gene expression. Ca2+-dependent gene expression is a major pathway controlled by the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), which in turn is induced by the thyroid hormone T3, as also demonstrated in a mouse embryonic stem cell line. In addition, T3 controls the expression of neurexin, synaptotagmin2 (SYT2), synaptotagmin-related gene1 (SRG1), and a number of other genes involved in neurotransmitter release in a Ca2+-dependent manner. It has been noticed that the development of dopaminergic neurons by evoking significant calcium entry occurs through TRPC calcium channels. It was also demonstrated that the T3-mediated development of an early neuronal network is characteristic for depolarizing GABAergic neurons concomitant with intracellular calcium transients. An important aspect of T3-dependent regulation of gene expression in the developing brain is its modulation by the transcription activator COUP-TF1. Regulation of alternative splicing by CaMKIV is another important aspect for embryonal neural development since it can lead to the expression of PMCA1a, the neuronal-specific isoform of the plasma membrane calcium pump. Maternal hypothyroidism or CaMKIV deficiency can have a severe influence on fetal brain development.

Thyroid hormones influence brain development through the regulation of gene expression. Ca

Calcium carries messages to virtually all important functions of cells. Although it was already active in unicellular organisms, its role became universally important after the transition to multicellular life. In this Minireview, we explore how calcium ended up in this privileged position. Most likely its unique coordination chemistry was a decisive factor as it makes its binding by complex molecules particularly easy even in the presence of large excesses of other cations, e.g. magnesium. Its free concentration within cells can thus be maintained at the very low levels demanded by the signaling function. A large cadre of proteins has evolved to bind or transport calcium. They all contribute to buffer it within cells, but a number of them also decode its message for the benefit of the target. The most important of these “calcium sensors” are the EF-hand proteins. Calcium is an ambivalent messenger. Although essential to the correct functioning of cell processes, if not carefully controlled spatially and temporally within cells, it generates variously severe cell dysfunctions, and even cell death.

In this chapter the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to the structural and functional properties of PMCA, the tissue distribution of the different isoforms, including their differences during development. The importance of PMCA for regulating Ca

Thyroid hormones influence brain development through regulation of gene expression. This is especially true for Ca 2 + -dependent regulation since a major pathway is controlled by the Ca 2 + /calmodulin-dependent protein kinase IV (CaMKIV) which in turn is induced by the thyroid hormone T 3 . In addition, CaMKIV is involved in regulation of alternative splicing of a number of protein isoforms, among them PMCA1a, the neuronal specific isoform of the plasma membrane calcium pump. On the other hand, hypothyroidism or CaMKIV deficiency can have a severe influence on brain development. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

In this chapter the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to the structural and functional properties of PMCA, the tissue distribution of the different isoforms, including their differences during development. The importance of PMCA for regulating Ca2+ signaling in microdomains under different conditions is also discussed.

publisher: Elsevier articletitle: “Ca2+ signaling and cell death”: The ECS 2013 workshop in Leuven and a tribute to Humbert De Smedt journaltitle: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research articlelink: http://dx.doi.org/10.1016/j.bbamcr.2014.05.015 content_type: simple-article copyright: Copyright © 2014 Elsevier B.V. All rights reserved. ispartof: Biochimica et Biophysica Acta. Molecular Cell Research vol:1843 issue:10 pages:2139-42 ispartof: location:Netherlands status: published

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca2+-signaling protein ALG-2 and the RNA-binding protein RBM22.

Cellular Ca(2+) homeostasis is maintained through the integrated and coordinated function of Ca(2+) transport molecules, Ca(2+) buffers and sensors. These molecules are associated with the plasma membrane and different cellular compartments, such as the cytoplasm, nucleus, mitochondria, and cellular reticular network, including the endoplasmic reticulum (ER) to control free and bound Ca(2+) levels in all parts of the cell. Loss of nutrients/energy leads to the loss of cellular homeostasis and disruption of Ca(2+) signaling in both the reticular network and cytoplasmic compartments. As an integral part of cellular physiology and pathology, this leads to activation of ER stress coping responses, such as the unfolded protein response (UPR), and mobilization of pathways to regain ER homeostasis.

Calcium may have a static, structure-stabilizing role in biological organs like the bones and the teeth, or may fulfill a dynamic function in cells as a regulator of signal-transduction pathways. This is made possible by the properties of the Ca2+ ion (e.g., high dehydration rate, great flexibility in coordinating ligands, largely irregular geometry of the coordination sphere). Since Ca2+ is a universal carrier of signals, the control of its homeostasis is of central importance for the organism. It involves exchanges between the skeleton (which is the major calcium reservoir) and the extracellular and intracellular fluids. It also involves the intestine and the kidney, the organs of Ca absorption and release, respectively. The highly integrated homeostasis process consists of a number of hormonally controlled feedback loops, and an elaborate system of membrane channels, exchangers, and pumps that control the Ca2+ flux into and out of cells.

This article describes the theories and practices of the German installation artists and composers Sabine Schäfer and Joachim Krebs. Much of their work concerns site-specific sound installations involving the articulation of time and space. Their principal work methods and materials are described. In addition, they have formulated a typology of five installation types which they describe using their own installations as examples. Each installation type responds to a particular set of aesthetic and practical challenges both for the artists and the visitors. These are discussed and illustrated in the article. The typology extends beyond the specific work of these artists and can be applied to installations in general, thus providing a framework for critical analysis. Furthermore, the translators have discussed the issues regarding the specialised vocabulary of the artists and the rendering of such language into English.

During the development of an organism, cell proliferation, differentiation and cell death are tightly balanced, and are controlled by a number of different regulators. Alterations in this balance are often observed in a variety of human diseases. The role of Ca(2+) as one of the key regulators of the cell is discussed with respect to a recently discovered Ca(2+)-binding protein, ALG-2, which is highly upregulated in cancerous tissues of different origins. The role of ALG-2 as a possible clinical marker and, molecularly, as a possible modulator at the interface between cell proliferation and cell death is discussed.

The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].

In this chapter various aspects of apoptosis or programmed cell death (PCD) influenced by calcium as a mediator of signal transduction have been reviewed. Attention has been focused on recently described calcium-binding proteins such as ALG-2 or on a new calcium/calmodulin-dependent kinase, the death associated protein kinase or DAP-kinase. Both play a central role in apoptotic processes. Calcineurin, which normally is involved in the regulation of T-cell proliferation, is reported to interact with the apoptosis protection protein bcl-2. Its possible involvement in the decision process whether T-cell activation leads to proliferation or apoptosis is discussed.

Calcium is a very versatile second messenger involved in the regulation of a variety of different cellular processes. This is made possible due to the binding of Ca 2+ to a great variety of different calcium-binding proteins, such as calcium-buffering or calcium-sensing proteins; the latter are also known as EF-hand containing proteins. In addition, a number of other calcium-binding proteins have been studied in recent years. In this article the families of annexins, gelsolins, and C 2 -domain containing proteins are described. These proteins are composed of a number of repeat units containing a variety of different Ca 2+ -binding sites. They bind to membranous or cytoskeletal structures in a Ca 2+ -dependent manner, and are involved in a number of different cellular functions.

The expression of calmodulin kinase IV (CaMKIV) can be induced by the thyroid hormone T3 in a time- and concentration-dependent manner at a very early state of brain differentiation using a fetal rat telencephalon primary cell culture system which can grow and differentiate under chemically defined conditions (Krebs et al. (1996) J. Biol. Chem. 271, 11055–11058). After the induction of CaMKIV by T3 we examined the influence of prolonged absence of T3 from the culture medium on the expression of CaMKIV. We could demonstrate that after the T3-dependent induction of CaMKIV, omission of the hormone, even for 8 days, from the medium did not downregulate the expression of CaMKIV indicating that different regulatory mechanisms became important for the expression of the enzyme. We further showed that CaMKIV could be involved in the Ca2+-dependent expression of the immediate early gene c-fos, probably via phosphorylation of the transcription factor CREB. Convergence of signal transduction pathways on this transcription factor by using different protein kinases may explain the importance of CREB for the regulation of different cellular processes.

The structure of the apo form of calcyclin, a member of the S100 family of calcium-binding proteins, reveals a novel dimer fold that may reflect the presence of a new interface for target protein recognition.

Mit diesem Kapitel beginnt der empirische Teil der vorliegenden Untersuchung, der sich in vier Sektionen unterteilen lasst. Dabei werden im unmittelbaren Anschluss zunachst die Datenerhebungs- und –analysemethoden vorgestellt, bevor sich die nachfolgenden Kapitel mit den Datenauswertungen anhand verschiedener statistischer Verfahren beschaftigen (vgl. Abbildung 16).

Das folgende Kapitel stellt empirische Ergebnisse vor, die erste Indikationen fur die Beantwortung der in Unterkapitel 1.2 formulierten Forschungsfragen und Belege fur die einzelnen in Unterkapitel 3.6 zusammengefassten Hypothesen liefern.

Dieses Kapitel reflektiert die empirischen Ergebnisse einer gesamthaften Betrachtung aller relevanten Einflussfaktoren des Beteiligungserfolgs, die eine Beurteilung des Forschungsmodells, Bewertung der Hypothesen und Beantwortung der Forschungsfragen ermoglicht. Zunachst wird in Unterkapitel 6.1 die Datenbasis der multivariaten Analysen beschrieben, und Unterschiede zur Grundlage der bivariaten Untersuchungen werden aufgezeigt.

Nachdem in Kapitel 2 das zum Verstandnis der vorliegenden Arbeit erforderliche Fundament gelegt worden ist, formuliert Kapitel 3 Hypothesen zu den Zusammenhangen zwischen wesentlichen Syndizierungsparametern bzw.

Ca(2+)-signaling, alternative splicing, and stress responses by the endoplasmic reticulum are three important cellular activities which can be strongly interconnected to alter the expression of protein isoforms in a tissue dependent manner or during development depending on the environmental conditions. This integrated network of signaling pathways permits a high degree of versatility and adaptation to metabolic, developmental and stress processes. Defects in its regulation may lead to cellular malfunction.

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.

ALG-2 is a highly conserved calcium binding protein in the cytoplasm which belongs to the family of penta-EF hand proteins. Recently, we showed that ALG-2 is interacting with RBM22, a highly conserved spliceosomal nuclear protein (Montaville et al. Biochim. Biophys. Acta 1763, 1335, 2006; Krebs, Biochim. Biophys. Acta 1793, 979, 2009). In NIH 3T3 cells expressing both proteins a significant amount of ALG-2mRFP is translocated to the nucleus due to the interaction with RBM22-EGFP. hSlu7, another spliceosomal nuclear protein, known to interact with RBM22 in yeast, has been shown to translocate to the cytoplasm under cellular stress conditions. Here we provide evidence that the 2 spliceosomal proteins differ significantly in their subcellular distributions under stress conditions, and that RBM22 enhances the cytoplasmic translocation of hSlu7 under stress, especially a stress induced by thapsigargin. On the other hand, in NIH 3T3 cells expressing RBM22-EGFP and ALG-2-mRFP, ALG-2 remains translocated into the nucleus under both stress conditions, i.e. heat shock or treatment with thapsigargin. We could further demonstrate that these stress conditions had a different influence on the splicing pattern of XBP-1, a marker for the unfolded protein response indicating that ER stress may play a role in stress-induced translocation of spliceosomal proteins. The article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).

In this review the influence of calcium signaling on the regulation of alternative splicing is discussed with respect to its influence on cell- and developmental-specific expression of different isoforms of the plasma membrane calcium pump (PMCA). In a second part the possibility is discussed that due to the interaction of the calcium-binding protein ALG-2 with a spliceosomal regulator of alternative splicing, RBM22, Ca2+-signaling may thus influence its regulatory property.

The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids. The experiments have shown that the binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-binding peptides.

Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with the calmodulin binding domain, supporting the suggestion (Benaim et al., 1984) that the latter, or a portion of it, may act as a natural inhibitor of the pump.

Calcium is of pivotal importance for many biological processes. It may have a rather static, structure-stabilizing role, or it may participate as one of the second messengers of the cell in signal transduction pathways, fulfilling a more dynamic function. This is made possible by some specific properties of the Ca 2+ ion (e.g. high dehydration rate, great flexibility in coordinating ligands and largely irregular geometry of the coordination sphere). The control of calcium homeostasis is of central importance for the organism. It is a highly integrated process consisting of a number of hormonally controlled feedback loops and an elaborate system of channels, exchangers, pumps and numerous Ca 2+ -binding proteins to control Ca 2+ fluxes into and out of cells or within cells. This chapter describes the different roles of calcium in the regulation of biological functions and the proteins involved in these processes.