EF-hand Calcium-binding Protein Research Articles

N-Methyl-D-Aspartate Receptor Link to the MAP Kinase Pathway in Cortical and Hippocampal Neurons and Microglia Is Dependent on Calcium Sensors and Is Blocked by α-Synuclein, Tau, and Phospho-Tau in Non-transgenic and Transgenic APPSw,Ind Mice.

N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APPSw,Ind transgenic Alzheimer’s disease model. Interestingly, a very marked increase in NMDAR–NCS1 complexes was identified in neurons and a marked increase of both NMDAR–NCS1 and NMDAR–CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor–calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APPSw,Ind mouse embryos/pups.

Comprehensive analysis of the roles of ‘black’ and ‘gray’ clusters in structure and function of rat β-parvalbumin

Recently we found two highly conserved structural motifs in the proteins of the EF-hand calcium binding protein family. These motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif forms a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is much more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II includes a mix of aromatic, hydrophobic, and polar amino acids. The ‘black’ and ‘gray’ clusters in rat β-parvalbumin consist of F48, A100, F103 and G61, L64, M87, respectively. In the present work, we sequentially substituted these amino acids residues by Ala, except Ala100, which was substituted by Val. Physical properties of the mutants were studied by circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probe methods. The Ca2+ and Mg2+ binding affinities of these mutants were evaluated by intrinsic fluorescence and equilibrium dialysis methods. In spite of a rather complicated pattern of contributions of separate amino acid residues of the ‘black’ and ‘gray’ clusters into maintenance of rat β-parvalbumin structural and functional status, the alanine substitutions in the cluster I cause noticeably more pronounced changes in various structural parameters of proteins, such as hydrodynamic radius of apo-form, thermal stability of Ca2+/Mg2+-loaded forms, and total energy of Ca2+ binding in comparison with the changes caused by amino acid substitutions in the cluster II. These findings were further supported by the outputs of computational analysis of the effects of these mutations on the intrinsic disorder predisposition of rat β-parvalbumin, which also indicated that local intrinsic disorder propensities and the overall levels of predicted disorder were strongly affected by mutations in the cluster I, whereas mutations in cluster II had less pronounced effects. These results demonstrate that amino acids of the cluster I provide more essential contribution to the maintenance of structuraland functional properties of the protein in comparison with the residues of the cluster II.

Oncomodulin Expression Reveals New Insights into the Cellular Organization of the Murine Utricle Striola.

Oncomodulin (OCM, aka β-parvalbumin) is an EF-hand calcium binding protein that is expressed in a restricted set of hair cells in the peristriolar region of the mammalian utricle. In the present study, we determined the topologic distribution of OCM among hair cell phenotypes to advance our understanding of the cellular organization of the striola and the relationship of these phenotypes with characteristics of tissue polarity. The distributions of OCM-positive (OCM+) hair cells were quantified in utricles of mature C57Bl/6 mice. Immunohistochemistry was conducted using antibodies to OCM, calretinin, and β3-tubulin. Fluorophore-conjugated phalloidin was used to label hair cell stereocilia, which provided the basis for determining hair cell counts and morphologic polarizations. We found OCM expression in striolar types I and II hair cells, though the distributions were dissimilar to the native striolar type I and II distributions, favoring type I hair cells. The distribution of OCM immunoreactivity among striolar type I hair cells also reflected nonrandom distribution among type Ic and Id phenotypes (i.e., those receiving calretinin-positive and calretinin-negative calyces, respectively). However, many OCM+ hair cells were found lateral to the striola, and within the epithelial region encompassing OCM+ hair cells, the distributions of OCM+ types Ic and Id hair cells were similar to the native distributions of Ic and Id in this region. Summarily, these data provide a quantitative perspective supporting the existence of different underlying factors driving the topologic expression of OCM in hair cells than those responsible for tissue polarity characteristics associated within the utricular striola, including calretinin expression in afferent calyces.

Identification of drought-inducible regulatory factors in Lablab purpureus by a comparative genomic approach

Physiological indices of two lines of hyacinth bean (Lablab purpureus (L.) Sweet), MEIDOU 2012 and NANHUI 23, were compared during a progressive course of water-withholding treatments. MEIDOU 2012 was shown to be a superior drought-tolerant genotype. A suppressive subtractive hybridisation library was then constructed by using drought-stressed MEIDOU 2012 roots and sequenced to screen for transcripts with differential abundance variations. In total, 2792 unigenes were assembled from 4064 drought-induced expressed sequence tags. Comparative analysis with other legume genomic sequences, including soybean (Glycine max (L.) Merr.), common bean (Phaseolus vulgaris L.) and lucerne (Medicago sativa L.), was performed, from which 338 unigenes associated with root growth and drought response were identified. Among these unigenes, 27 transcription factors were deduced by functional-domain prediction. The transcription levels of a GRAS and a WRKY transcription factor were confirmed to be responding sensitively to water stresses. Additionally, protein–protein interaction (PPI) networks were predicted for all drought-induced root-related unigenes with different stringency levels. A mitogen-activated protein kinase (MAPK4) and an EF-hand calcium-binding protein (CML24) module were pinpointed to be putative ‘master’ signalling hubs in these PPI networks. Detailed examination of these genomic resources would further reveal key regulators of Lablab drought tolerance.

Developmental biology and potential use of Alboglossiphonia lata (Annelida: Hirudinea) as an \u201cEvo-Devo\u201d model organism

BackgroundThe need for the adaptation of species of annelids as “Evo-Devo” model organisms of the superphylum Lophotrochozoa to refine the understanding of the phylogenetic relationships between bilaterian organisms, has promoted an increase in the studies dealing with embryonic development among related species such as leeches from the Glossiphoniidae family. The present study aims to describe the embryogenesis of Alboglossiphonia lata (Oka, 1910), a freshwater glossiphoniid leech, chiefly distributed in East Asia, and validate standard molecular biology techniques to support the use of this species as an additional model for “Evo-Devo” studies.ResultsA. lata undergoes direct development, and follows the highly conserved clitellate annelid mode of spiral cleavage development; the duration from the egg laying to the juvenile stage is ~7.5 days, and it is iteroparous, indicating that it feeds and deposits eggs again after the first round of brooding, as described in several other glossiphoniid leech species studied to date. The embryos hatch only after complete organ development and proboscis retraction, which has not yet been observed in other glossiphoniid genera. The phylogenetic position of A. lata within the Glossiphoniidae family has been confirmed using cytochrome c oxidase subunit 1 (CO1) sequencing. Lineage tracer injections confirmed the fates of the presumptive meso- and ectodermal precursors, and immunostaining showed the formation of the ventral nerve system during later stages of development. Further, the spatiotemporal expression of an EF-hand calcium-binding protein Calsensin ortholog was characterized, which showed a specific pattern in both the ventral and peripheral nervous systems during the later stages.ConclusionsOur description of the embryonic development of A. lata under laboratory conditions provides new data for further comparative studies with other leech and lophotrochozoa model organisms. Moreover, it offers a basis for the establishment of this species as a model for future “Evo-Devo” studies.

In search for globally disordered apo-parvalbumins: Case of parvalbumin β-1 from coho salmon

Parvalbumin (PA) is a classical EF-hand calcium-binding protein of muscle, neuronal, and other tissues, and a major fish allergen. Although certain apo-PAs lack tertiary structure, functional implications of that feature and its structural prerequisites remain unclear. In a search for unstable PAs, we probed conformational stability of parvalbumin β-1 from coho salmon (csPA), a cold water fish species, using circular dichroism, scanning calorimetry, hydrophobic probe fluorescence, limited proteolysis, chemical crosslinking and dynamic light scattering techniques. Apo-csPA is shown to be mainly monomeric protein with markedly disorganized secondary structure and lack of rigid tertiary structure. Examination of per-residue propensity for intrinsic disorder in the PA groups with either folded or unfolded apo-form using the average PONDR® VSL2P profiles revealed that the N-terminal region that includes α-helix A, AB-loop and N-terminal half of α-helix B is predicted to be less ordered in PAs with disordered apo-state. Application of the structural criteria developed for discrimination of disordered PAs indicate that the latter comprise about 16–19% of all PAs. We show that structural instability of apo-β-PA serves as a hallmark of elevated calcium affinity of the protein. Therefore, the successful predictions of unstable apo-PAs might facilitate search for PAs with maximal calcium affinity and possibly serving as calcium sensors.

Ca2+ Binding and Conformational Switch of the Photoprotein Mnemiopsin.

Bioluminescence in Ca2+-binding photoproteins is an intramolecular reaction triggered by the addition of Ca2+. A comparative study has been done on Ca2+-depleted and Ca2+-loaded apo-mnemiopsin to understand the structural transition of the photoprotein by Ca2+ binding. Ca2+ is removed by TCA (trichloroacetic acid) precipitation to obtain Ca2+-depleted apomnemiopsin. UV-visible, CD and fluorescence spectroscopic studies demonstrate that the addition of Ca2+ is brought about by the overall structure of apo-mnemiopsin becomes more open in a concentration- dependent manner without significantly influencing the secondary structure and indicate that the Ca2+-depleted form of apo-mnemiopsin, in contrast to most other EF-hand calcium binding proteins, adopt a closed conformation when compared to the Ca2+-loaded form. On the other hand, dynamic quenching and limited proteolysis analysis revealed that Ca2+-loaded apo-mnemiopsin became much more flexible than Ca2+ free apo-mnemiopsin. It seems that increased flexibility of the protein, which occurs due to calcium binding, is a critical factor in oxidative decarboxylation reaction on coelenterazine and consequently light emission.

Calcium Ion Induced Structural Changes Promote Dimerization of Secretagogin, Which Is Required for Its Insulin Secretory Function

Secretagogin (SCGN), a hexa EF-hand calcium binding protein, plays key roles in insulin secretion in pancreatic β-cells. It is not yet understood how the binding of Ca2+ to human SCGN (hSCGN) promotes secretion. Here we have addressed this question, using mass spectrometry combined with a disulfide searching algorithm DBond. We found that the binding of Ca2+ to hSCGN promotes the dimerization of hSCGN via the formation of a Cys193-Cys193 disulfide bond. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics studies revealed that Ca2+ binding to the EF-hands of hSCGN induces significant structural changes that affect the solvent exposure of N-terminal region, and hence the redox sensitivity of the Cys193 residue. These redox sensitivity changes were confirmed using biotinylated methyl-3-nitro-4-(piperidin-1-ylsulfonyl) benzoate (NPSB-B), a chemical probe that specifically labels reactive cysteine sulfhydryls. Furthermore, we found that wild type hSCGN overexpression promotes insulin secretion in pancreatic β cells, while C193S-hSCGN inhibits it. These findings suggest that insulin secretion in pancreatic cells is regulated by Ca2+ and ROS signaling through Ca2+-induced structural changes promoting dimerization of hSCGN.

S100A12-CD36 axis: A novel player in the pathogenesis of atherosclerosis?

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins and have a variety of intracellular and extracellular activities. It exerts its proinflammatory effects by binding to the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4). CD36 is a class B scavenger receptor that acts as a fatty acid transporter. Both S100A12 and CD36 are implicated in vascular inflammation and atherosclerosis. It has recently been demonstrated that S100A12 binds with high affinity to CD36. On the other hand, RAGE and TLR4 play a key role in the regulation of CD36 expression. These observations point to the fact that S100A12 is an interesting molecular target for the development of therapeutics. This Cytokine stimulus will focus on the possible mechanisms of S100A12-CD36 axis in the pathogenesis of atherosclerosis.

Functional expression, characterization, and application of human S100B.

The EF-hand calcium-binding protein S100B presents a wide range of biological activities and functions. This binding protein is involved in various human diseases, including cancer, brain trauma and ischemia, neuro-degenerative disease (Alzheimer's disease), and psychiatric disorders. In this study, we prepared human S100B protein and its monoclonal antibodies. Human S100B protein was expressed in Escherichia coli, successfully purified by diethylaminoethyl cellulose anion-exchange chromatography, and then identified by western blot analysis. Monoclonal antibodies (mAbs) were produced by the standard hybridoma method and validated by enzyme-linked immunosorbent assay and western blot analysis. The prepared human S100B protein and its mAbs demonstrated potential biological activities. The KD of one mAb is approximately 4.72x10-8mol/l, and its cross reactivity is low with human S100A4, mouse S100A4, and human S100A1. Recombinant Soluble S100B can promote the migration and invasion of HeLa cells. The expression of S100B protein in tumor tissues can be detected effectively by using the prepared monoclonal antibodies. Increasing concentration of the anti-human S100B mAbs showed a reduced expression of the S100B protein. Subsequently, the expression of p53 increased significantly (P<0.05) in A375 cells. A significant increase in apoptosis in A375 cells was observed with increasing S100B mAb concentration. Results showed that our prepared S100B mAbs were suitable for detecting S100B expression in human tissues, furnishing promising tools for further functional investigation and clinical applications.

S100A7 promotes the migration, invasion and metastasis of human cervical cancer cells through epithelial-mesenchymal transition.

S100A7 is an EF-hand calcium-binding protein that has been suggested to be implicated in cell proliferation, migration, invasion and tumor metastasis. However, its role in cervical cancer has not yet been fully clarified. The present study used immunohistochemistry analysis of S100A7 in clinical specimens of cervical cancer to show that S100A7 expression was significantly upregulated in cervical cancer tissues compared with normal cervical tissues and S100A7 expression in high grade cervical intraepithelial neoplasm (CIN) was significantly higher than cervical cancer. Statistical analysis showed that S100A7 expression was associated with tumor grade (P <0.01) and lymph node metastasis (P <0.05). Functional studies showed that overexpression of S100A7 in cervical cancer cells promoted migration, invasion and metastasis of cervical cancer cells without influencing cell proliferation. Furthermore, S100A7 was found to be secreted into the conditioned media and extracellular S100A7 enhanced cell migration and invasion. Mechanistically, S100A7 bound to RAGE and activated ERK signaling pathway. And S100A7 enhanced cell mesenchymal properties and induced epithelial–mesenchymal transition. In summary, these data reveal a crucial role for S100A7 in regulating cell migration, invasion, metastasis and EMT of cervical cancer and suggest that targeting S100A7 may offer a new targeted strategy for cervical cancer.

A salivary EF-hand calcium-binding protein of the brown planthopper Nilaparvata lugens functions as an effector for defense responses in rice

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a major pest of rice in Asia, is able to successfully puncture sieve tubes in rice with its piercing stylet and then to ingest phloem sap. How BPH manages to continuously feed on rice remains unclear. Here, we cloned the gene NlSEF1, which is highly expressed in the salivary glands of BPH. The NlSEF1 protein has EF-hand Ca2+-binding activity and can be secreted into rice plants when BPH feed. Infestation of rice by BPH nymphs whose NlSEF1 was knocked down elicited higher levels of Ca2+ and H2O2 but not jasmonic acid, jasmonoyl-isoleucine (JA-Ile) and SA in rice than did infestation by control nymphs; Consistently, wounding plus the recombination protein NlSEF1 suppressed the production of H2O2 in rice. Bioassays revealed that NlSEF1-knockdown BPH nymphs had a higher mortality rate and lower feeding capacity on rice than control nymphs. These results indicate that the salivary protein in BPH, NlSEF1, functions as an effector and plays important roles in interactions between BPH and rice by mediating the plant’s defense responses.

Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate Euplotes octocarinatus centrin.

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca2+ and Tb3+) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37>Asp146>Asp110>Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.

Insights into binding of S100 proteins to scavenger receptors: class B scavenger receptor CD36 binds S100A12 with high affinity

The EF-hand type calcium-binding protein S100A12 exerts numerous intra- and extracellular functions of (patho)physiological relevance. Therefore, receptors of S100A12 are of high interest for research and clinical applications. Beside the extensively studied receptor for advanced glycation endproducts (RAGE), G-protein coupled receptors and more recently, scavenger receptors are suggested to be putative S100A12 receptors. Own findings and further information from the literature predestined CD36, a class B scavenger receptor, as promising candidate. To substantiate or prove against this hypothesis, this study aimed at investigation of interaction of S100A12 and CD36 on molecular and cellular level by the use of surface plasmon resonance (SPR), radio- and fluorescence-tracer-based cell binding, and cell activation experiments. S100A12 revealed binding affinity to CD36 in the low nanomolar range, essentially, at the CD36 thrombospondin-1 binding site. Additionally, S100A12-mediated translocation of CD36 to the membrane and elevation of both CD36 and peroxisome proliferator-activated receptor γ (PPARγ) expression was observed, which suggest a potential regulatory function of S100A12–CD36 interaction.

Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

NECAB3 Promotes Activation of Hypoxia-inducible factor-1 during Normoxia and Enhances Tumourigenicity of Cancer Cells.

Unlike most cells, cancer cells activate hypoxia inducible factor-1 (HIF-1) to use glycolysis even at normal oxygen levels, or normoxia. Therefore, HIF-1 is an attractive target in cancer therapy. However, the regulation of HIF-1 during normoxia is not well characterised, although Mint3 was recently found to activate HIF-1 in cancer cells and macrophages by suppressing the HIF-1 inhibitor, factor inhibiting HIF-1 (FIH-1). In this study, we analysed Mint3-binding proteins to investigate the mechanism by which Mint3 regulates HIF-1. Yeast two-hybrid screening using Mint3 as bait identified N-terminal EF-hand calcium binding protein 3 (NECAB3) as a novel factor regulating HIF-1 activity via Mint3. NECAB3 bound to the phosphotyrosine-binding domain of Mint3, formed a ternary complex with Mint3 and FIH-1, and co-localised with Mint3 at the Golgi apparatus. Depletion of NECAB3 decreased the expression of HIF-1 target genes and reduced glycolysis in normoxic cancer cells. NECAB3 mutants that binds Mint3 but lacks an intact monooxygenase domain also inhibited HIF-1 activation. Inhibition of NECAB3 in cancer cells by either expressing shRNAs or generating a dominant negative mutant reduced tumourigenicity. Taken together, the data indicate that NECAB3 is a promising new target for cancer therapy.

Centrin 4 is a Binding Partner of Rat EAG1 K+ Channels

The ether-a-go-go (Eag) potassium (K+) channel belongs to the superfamily of voltage-gated K+ channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was centrin 4, which is an EF-hand calcium-binding protein with four putative calciumion-binding sites. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with centrin was mediated by the carboxyl-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and brain lysates. Centrin 4 and calmodulin are closely related four-EF-hand calcium-binding proteins. The binding affinity of calmodulin to rEag1 was notably enhanced in the presence of calcium. In contrast, the interaction between centrin 4 and rEag1 appeared to be calcium-independent. We therefore propose that centrin 4 may serve as a constitutive binding-partner of rEag1.

S100A7: A rAMPing up AMP molecule in psoriasis

S100A7 (psoriasin), an EF-hand type calcium binding protein localized in epithelial cells, regulates cell proliferation and differentiation. An S100A7 overexpression may occur in response to inflammatory stimuli, such in psoriasis, a chronic inflammatory autoimmune-mediated skin disease. Increasing evidence suggests that S100A7 plays critical roles in amplifying the inflammatory process in psoriatic skin, perpetuating the disease phenotype. This review will discuss the interactions between S100A7 and cytokines in psoriatic skin. Furthermore, we will focus our discussion on regulation and functions of S100A7 in psoriasis. Finally, we will discuss the possible use of S100A7 as therapeutic target in psoriasis.

Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.

Synaptic Targets of Medial Septal Projections in the Hippocampus and Extrahippocampal Cortices of the Mouse.

Temporal coordination of neuronal assemblies among cortical areas is essential for behavioral performance. GABAergic projections from the medial septum and diagonal band complex exclusively innervate GABAergic interneurons in the rat hippocampus, contributing to the coordination of neuronal activity, including the generation of theta oscillations. Much less is known about the synaptic target neurons outside the hippocampus. To reveal the contribution of synaptic circuits involving the medial septum of mice, we have identified postsynaptic cortical neurons in wild-type and parvalbumin-Cre knock-in mice. Anterograde axonal tracing from the septum revealed extensive innervation of the hippocampus as well as the subiculum, presubiculum, parasubiculum, the medial and lateral entorhinal cortices, and the retrosplenial cortex. In all examined cortical regions, many septal GABAergic boutons were in close apposition to somata or dendrites immunopositive for interneuron cell-type molecular markers, such as parvalbumin, calbindin, calretinin, N-terminal EF-hand calcium-binding protein 1, cholecystokinin, reelin, or a combination of these molecules. Electron microscopic observations revealed septal boutons forming axosomatic or axodendritic type II synapses. In the CA1 region of hippocampus, septal GABAergic projections exclusively targeted interneurons. In the retrosplenial cortex, 93% of identified postsynaptic targets belonged to interneurons and the rest to pyramidal cells. These results suggest that the GABAergic innervation from the medial septum and diagonal band complex contributes to temporal coordination of neuronal activity via several types of cortical GABAergic interneurons in both hippocampal and extrahippocampal cortices. Oscillatory septal neuronal firing at delta, theta, and gamma frequencies may phase interneuron activity.