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Neuronal calcium sensor 1 (NCS1) regulates a wide range of cellular functions throughout the mammalian nervous systems. Altered NCS1 expression is associated with neurodevelopmental and neurodegenerative diseases. Previous studies focused on affective and cognitive behaviors in NCS1 knockout (KO) mice, but little is known about the physiological and pathological states associated with the loss of NCS1 in the peripheral nervous system. We previously reported that NCS1 expression was reduced following paclitaxel-induced peripheral neuropathy. Here, we comprehensively investigated the phenotypes of NCS1-KO mice through a battery of behavioral tests examining both central and peripheral nervous systems. Generally, only mild differences were observed in thermal sensation and memory acquisition between NCS1-WT and -KO male mice, but not in female mice. No differences were observed in motor performance, affective behaviors, and hearing in both sexes. These results suggest that NCS1 plays a modulatory role in sensory perceptions and cognition, particularly in male mice. NCS1 has been proposed as a pharmacological target for various diseases. Therefore, the sex-specific effects of NCS1 loss may be of clinical interest. As we examined a constitutive KO model, future studies focusing on various conditional KO models will further elucidate the precise physiological significance of NCS1.

Changes in intracellular calcium (Ca2+) signaling can modulate cellular machinery required for cancer progression. Neuronal calcium sensor 1 (NCS1) is a ubiquitously expressed Ca2+‐binding protein that promotes tumor aggressiveness by enhancing cell survival and metastasis. However, the underlying mechanism by which NCS1 contributes to increased tumor aggressiveness has yet to be identified. In this study, we aimed to determine (a) whether NCS1 expression changes in response to external stimuli, (b) the importance of NCS1 for cell survival and migration, and (c) the cellular mechanism(s) through which NSC1 modulates these outcomes. We found that NCS1 abundance increases under conditions of stress, most prominently after stimulation with the pro‐inflammatory cytokine tumor necrosis factor α, in a manner dependent on nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB). We found that NFκB signaling is activated in human breast cancer tissue, which was accompanied by an increase in NCS1 mRNA expression. Further exploration into the relevance of NCS1 in breast cancer progression showed that knockout of NCS1 (NCS1 KO) caused decreased cell survival and motility, increased baseline intracellular Ca2+ levels, and decreased inositol 1,4,5‐trisphosphate‐mediated Ca2+ responses. Protein kinase B (Akt) activity was decreased in NCS1 KO cells, which could be rescued by buffering intracellular Ca2+. Conversely, Akt activity was increased in cells overexpressing NCS1 (NCS1 OE). We therefore conclude that NCS1 acts as cellular stress response protein up‐regulated by stress‐induced NFκB signaling and that NCS1 influences cell survival and motility through effects on Ca2+ signaling and Akt pathway activation., This study investigates the underlying mechanism by which the calcium (Ca2+)‐binding protein NCS1 promotes increased tumor aggressiveness. We characterize NCS1 as a stress response protein that is up‐regulated by exogenous stressors, including the cytokine tumor necrosis factor α, through a nuclear factor kappa‐light‐chain‐enhancer of activated B cells‐dependent mechanism. Increased NCS1 expression leads to disrupted intracellular Ca2+ signaling and activation of the protein kinase B pathway, leading to increased cell survival and migration.

Peripheral biomarker and post-mortem brains studies have shown alterations of neuronal calcium sensor 1 (Ncs-1) expression in people with bipolar disorder or schizophrenia. However, its engagement by psychiatric medications and potential contribution to behavioral regulation remains elusive. We investigated the effect on Ncs-1 expression of valproic acid (VPA), a mood stabilizer used for the management of bipolar disorder. Treatment with VPA induced Ncs-1 gene expression in cell line while chronic administration of this drug to mice increased both Ncs-1 protein and mRNA levels in the mouse frontal cortex. Inhibition of histone deacetylases (HDACs), a known biochemical effect of VPA, did not alter the expression of Ncs-1. In contrast, pharmacological inhibition or genetic downregulation of glycogen synthase kinase 3β (Gsk3β) increased Ncs-1 expression, whereas overexpression of a constitutively active Gsk3β had the opposite effect. Moreover, adeno-associated virus-mediated Ncs-1 overexpression in mouse frontal cortex caused responses similar to those elicited by VPA or lithium in tests evaluating social and mood-related behaviors. These findings indicate that VPA increases frontal cortex Ncs-1 gene expression as a result of Gsk3 inhibition. Furthermore, behavioral changes induced by Ncs-1 overexpression support a contribution of this mechanism in the regulation of behavior by VPA and potentially other psychoactive medications inhibiting Gsk3 activity.

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum–localized channels that mediate Ca(2+) release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca(2+)-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66–110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1–InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca(2+) signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca(2+) signaling and survival, further suggesting the importance of this residue in the NCS1–InsP3R1 interaction. In conclusion, we show that NCS1–InsP3R1 interaction enhances intracellular Ca(2+) signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1–InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.

Calcium (Ca2+) signaling is critical for neuronal functioning and requires the concerted interplay of numerous Ca2+-binding proteins, including neuronal calcium sensor 1 (NCS1). Although an important role of NCS1 in neuronal processes and in neurodevelopmental and neurodegenerative diseases has been established, the underlying mechanisms remain enigmatic. Here, we systematically investigated the functions of NCS1 in the brain. Using Golgi-Cox staining, we observed a reduction in dendritic complexity and spine density in the prefrontal cortex and the dorsal hippocampus of Ncs1(−/−) mice, which may underlie concomitantly observed deficits in memory acquisition. Subsequent RNA sequencing of Ncs1(−/−) and Ncs1(+/+) mouse brain tissues revealed that NCS1 modulates gene expression related to neuronal morphology and development. Investigation of developmental databases further supported a molecular role of NCS1 during brain development by identifying temporal gene expression patterns. Collectively, this study provides insights into NCS1-dependent signaling and lays the foundation for a better understanding of NCS1-associated diseases.

Neuronal calcium sensor 1 (NCS1), a calcium-binding protein, and transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, are fundamental in the regulation of calcium homeostasis. The interactions of these proteins and their regulation by paclitaxel (PTX) were investigated using biochemical, pharmacological, and electrophysiological approaches in both a breast cancer epithelial cell model and a neuronal model. TRPV4 and NCS1 reciprocally immunoprecipitated each other, suggesting that they make up a signaling complex. The functional consequence of this physical association was that TRPV4 currents increased with increased NCS1 expression. Calcium fluxes through TRPV4 correlated with the magnitude of TRPV4 currents, and these calcium fluxes depended on NCS1 expression levels. Exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes but decreased the expression of NCS1. These findings augment the understanding of the properties of TRPV4, the role of NCS1 in the regulation of TRPV4, and the cellular mechanisms of PTX-induced neuropathy. SIGNIFICANCE STATEMENT TRPV4 and NCS1 physically and functionally interact. Increased expression of NCS1 enhances TRPV4-dependent currents, which are further amplified by treatment with the chemotherapeutic drug paclitaxel, an effect associated with adverse effects of chemotherapy, including neuropathy.

Funder: Lundbeckfonden; doi: http://dx.doi.org/10.13039/501100003554, Funder: Villum Fonden (DK), Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways.

Obesity associates with plethora of dysfunctions including changes in the brain structures, like hippocampus which is particularly susceptible to metabolic or inflammatory alterations. The is a need to expand knowledge about the obesity related brain changes in order to enable development of new therapeutic interventions, that would support not only weight loss but also could help to restore the after-effects of obesity in the brain. In the present report we evaluated the hippocampal efficiency of obese rats. In order to induce obesity 4 weeks old male rats were fed with a chow that mimics human western diet for 12 weeks (model of diet induced obesity). Then the cognitive functions (water maze), neurogenesis (BrdU detection) and global proteomic changes (MS/MS analysis) were analyzed in separate cohorts of animals. Obese rats had worsen cognitive flexibility than normal rats, but the spatial learning and long term memory were unaffected. The number of BrdU/NeuN positive cells was lowered in obese animals suggesting decreased hippocampal neurogenesis. The analysis of proteins revealed that about 120 proteins significantly differed between hippocampi of obese and normal rats. Neuronal calcium sensor 1 (NCS-1) which participates in mechanisms involving synaptic plasticity and acquisition of spatial memory was decreased in obese rats. Neuromodulin and vinculin, required for synaptic plasticity also decreased in these animals. Impaired neurogenesis was associated with decreased level of: receptor-type tyrosine-protein phosphatase zeta and cytoplasmic dynein 1 light intermediate chain 2, which play crucial roles in controlling axonal growth and neuronal migration. Altogether the data show that proteomic changes related to obesity associate with impaired hippocampal functioning. The same set of experiments was performed on the additional cohort of obese animals subsequent to weight loss to examine if the observed brain changes persist if obesity is reversed. This research is supported by Nutricia Foundation, grant no. RG 1/2017.

Electroacupuncture (EA) administration before or after cerebral ischemia has been shown to afford protection against ischemic injury. However, the underlying mechanism of EA-mediated protection is still unclear. Functional microRNAs (miRNAs) are believed to play important roles in neuroprotection and synaptic plasticity during and after ischemia. In a previous study, we identified 20 miRNAs that are expressed in the penumbra and are significantly changed after EA treatment. Here, we used bioinformatic analysis to predict the biological functions and gene networks of these miRNAs. Consistent with our predictions, downregulation of miR-191a-5p in primary neurons and in cortexes of rats increased cell viability, decreased apoptosis, reduced infarct volumes, and improved neurological scores; whereas upregulation of miR-191a-5p exacerbated neuronal injury and partly reversed the neuroprotective effect of EA treatment after ischemia/reperfusion injury. In silico analysis predicted that miR-191a-5p targets neuronal calcium sensor 1 (NCS-1), brain-derived neurotrophic factor, and growth-associated protein 43 (GAP43), and using luciferase reporter assays, we confirmed that the NCS-1 3'UTR (untranslated region) is targeted by miR-191a-5p. Furthermore, lentivirus-mediated overexpression of NCS-1 in primary neurons and in the cortexes of rats induced neuroprotection, while lentivirus-mediated knockdown had the opposite effect. Taken together, these data suggest that miRNAs participate in the response to EA treatment after cerebral ischemia and further imply that NCS-1 may constitute a miR-191a-5p target gene and a potential therapeutic target for neuroprotection.

Neuronal calcium sensor-1 (NCS-1) has been identified as a binding partner of the taxane, paclitaxel. Our previous study showed that overexpression of NCS-1 increased the efficacy of paclitaxel in vitro, but was associated with poor clinical outcome. Here, we determine if NCS-1 expression is associated with pathological complete response (pCR) to taxane-based neoadjuvant chemotherapy in 105 pre-treatment breast cancer biopsies. Elevated expression of NCS-1 was found to be positively associated with pCR. These results suggest that NCS-1 may be a predictive biomarker for response to taxane-based neoadjuvant chemotherapy in breast cancer.

Neuronal calcium sensor-1 (NCS-1) knockout (KO) in mice (NCS-1−/− mice) evokes behavioral phenotypes ranging from learning deficits to avolition and depressive-like behaviors. Here, we showed that with the onset of adulthood NCS-1−/− mice gain considerable weight. Adult NCS-1−/− mice are obese, especially when fed a high-fat diet (HFD), are hyperglycemic and hyperinsulinemic and thus develop a diabetes type 2 phenotype. In comparison to wild type (WT) NCS-1−/− mice display a significant increase in adipose tissue mass. NCS-1−/− adipocytes produce insufficient serum concentrations of resistin and adiponectin. In contrast to WT littermates, adipocytes of NCS-1−/− mice are incapable of up-regulating insulin receptor (IR) concentration in response to HFD. Thus, HFD-fed NCS-1−/− mice exhibit in comparison to WT littermates a significantly reduced IR expression, which may explain the pronounced insulin resistance observed especially with HFD-fed NCS-1−/− mice. We observed a direct correlation between NCS-1 and IR concentrations in the adipocyte membrane and that NCS-1 can be co-immunoprecipitated with IR indicating a direct interplay between NCS-1 and IR. We propose that NCS-1 plays an important role in adipocyte function and that NCS-1 deficiency gives rise to obesity and diabetes type 2 in adult mice. Given the association of altered NCS-1 expression with behaviorial abnormalities, NCS-1−/− mice may offer an interesting perspective for studying in a mouse model a potential genetic link between some psychiatric disorders and the risk of being obese.

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. The causes of PD in humans are still unknown, although metabolic characteristics of the neurons affected by the disease have been implicated in their selective susceptibility. Mitochondrial dysfunction and proteostatic stress are recognized to be important in the pathogenesis of both familial and sporadic PD, and they both culminate in bioenergetic deficits. Exposure to calcium overload has recently emerged as a key determinant, and pharmacological treatment that inhibits Ca2+ entry diminishes neuronal damage in chemical models of PD. In this review, we first introduce general concepts on neuronal Ca2+ signaling and then summarize the current knowledge on fundamental properties of substantia nigra pars compacta dopaminergic neurons, on the role of the interplay between Ca2+ and dopamine signaling in neuronal activity and susceptibility to cell death. We also discuss the possible involvement of a “neglected” player, the Neuronal Calcium Sensor-1 (NCS-1), which has been shown to participate to dopaminergic signaling by regulating dopamine dependent receptor desensitization in normal brain but, data supporting a direct role in PD pathogenesis are still missing. However, it is intriguing to speculate that the Ca2+-dependent modulation of NCS-1 activity could eventually counteract dopaminergic neurons degeneration.

The EF-hand calcium (Ca2+)-binding protein, neuronal Ca2+ sensor-1 (NCS-1/frequenin), is predominantly expressed in neuronal tissues and plays a crucial role in neuronal functions, including synaptic transmission and plasticity. NCS-1 has diverse functional roles, as elucidated in the past 15 years, which include the regulation of phosphatidylinositol 4-kinase IIIβ (PI-4K-β) and several ion channels such as voltage-gated K+ and Ca2+ channels, the D2 dopamine receptors, and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Functional analyses demonstrated that NCS-1 enhances exocytosis and neuronal survival after injury, as well as promotes learning and memory in mice. NCS-1 is also expressed in the heart including the Purkinje fibers (PFs) of the conduction system. NCS-1 interacts with KV4 K+ channels together with dipeptidyl peptidase-like protein-6 (DPP-6), and this macromolecule then composes the transient outward current in PFs and contributes to the repolarization of PF action potential, thus being responsible for idiopathic arrhythmia. Moreover, NCS-1 expression was reported to be significantly high at the immature stage and at hypertrophy in adults. That report demonstrated that NCS-1 positively regulates cardiac contraction in immature hearts by increasing intracellular Ca2+ signals through interaction with InsP3Rs. With the related signals, NCS-1 activates nuclear Ca2+ signals, which would be a mechanism underlying hormone-induced cardiac hypertrophy. Furthermore, NCS-1 contributes to stress tolerance in cardiomyocytes by activating mitochondrial detoxification pathways, with a key role in Ca2+-dependent pathways. In this review, we will discuss recent findings supporting the functional significance of NCS-1 in the brain and heart and will address possible underlying molecular mechanisms.

We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca2+ stress in Neurospora crassa. Increasing concentration of Ca2+ increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca2+ concentrations. These results suggested the regulation of NCS-1 function through calcineurin- CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that the CRZ-1 binds specifically to an 8 bp sequence 5'-CCTTCACA-3' in the ncs-1 promoter 216 bp upstream of the ATG start codon. We also showed that NCS-1 binds to the Ca2+ permeable channel MID-1 for tolerance to Ca2+ stress. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter, causing upregulation of NCS-1 that binds to MID-1 for tolerance to Ca2+ stress.

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca2+-bound, Mg2+-bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca2+ and Mg2+) to its EF-hands. At pathologically high Ca2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties.

Neuronal Calcium Sensor-1 (NCS1) belongs to the family of Neuronal Calcium Sensor (NCS) proteins. NCS1 is composed of four EF-hand motifs and an N-terminal myristoylation. However, the presence of a calcium-myristoyl switch in NCS1 and its role in the membrane binding are controversial. The model of Langmuir lipid monolayers is thus used to mimic the cell membrane in order to characterize the membrane interactions of NCS1. Two binding parameters are calculated from monolayer measurements: the maximum insertion pressure, up to which protein binding is energetically favorable, and the synergy, reporting attractive or repulsive interactions with the lipid monolayers. Binding membrane measurements performed in the presence of myristoylated NCS1 reveal better binding interactions for phospholipids composed of phosphoethanolamine polar head groups and unsaturated fatty acyl chains. In the absence of calcium, the membrane binding measurements are drastically modified and suggest that the protein is more strongly bound to the membrane. Indeed, the binding of calcium by three EF-hand motifs of NCS1 leads to a conformation change. NCS1 arrangement at the membrane could thus be reshuffled for better interactions with its substrates. The N-terminal peptide of NCS1 is composed of two amphiphilic helices involved in the membrane interactions of NCS1. Moreover, the presence of the myristoyl group has a weak influence on the membrane binding of NCS1 suggesting the absence of a calcium-myristoyl switch mechanism in this protein. The myristoylation could thus have a structural role required in the folding/unfolding of NCS1 which is essential to its multiple biological functions.

Calcium sensors detect intracellular calcium changes and interact with downstream targets to regulate many functions. Neuronal Calcium Sensor-1 (NCS-1) or Frequenin is widely expressed in the nervous system, and involved in neurotransmission, synaptic plasticity and learning. NCS-1 interacts with and regulates dopamine D2 receptor (D2R) internalization and is implicated in disorders like schizophrenia and substance abuse. However, the role of NCS-1 in behaviors dependent on dopamine signaling in the striatum, where D2R is most highly expressed, is unknown. We show that Ncs-1 deletion in the mouse decreases willingness to work for food. Moreover, Ncs-1 knockout mice have significantly lower activity-dependent dopamine release in the nucleus accumbens core in acute slice recordings. In contrast, food preference, responding for conditioned reinforcement, ability to represent changes in reward value, and locomotor response to amphetamine are not impaired. These studies identify novel roles for NCS-1 in regulating activity-dependent striatal dopamine release and aspects of motivated behavior.

Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks. * ACBD3, : acyl-CoA-binding domain containing protein 3; NCS-1, : neuronal calcium sensor 1; NS5A, : non-structural protein 5A; PI4K, : phosphatidylinositol 4 kinase; PI4KIIIα/β, : type III phosphatidylinositol 4 kinase α/β; PI4P, : phosphatidylinositol 4-phosphate; PIP2, : phosphatidylinositol 4,5-bisphosphate; PIP3, : phosphatidylinositol 3,4,5-trisphosphate; TGN, : trans -Golgi network; TTC7, : tetratricopeptide repeat protein 7

Neuronal calcium sensor-1 or Frequenin is a calcium sensor widely expressed in the nervous system, with roles in neurotransmission, neurite outgrowth, synaptic plasticity, learning, and motivated behaviours. Neuronal calcium sensor-1 has been implicated in neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and bipolar disorder. However, the role of neuronal calcium sensor-1 in behavioural phenotypes and brain changes relevant to autism spectrum disorder have not been evaluated. We show that neuronal calcium sensor-1 deletion in the mouse leads to a mild deficit in social approach and impaired displaced object recognition without affecting social interactions, behavioural flexibility, spatial reference memory, anxiety-like behaviour, or sensorimotor gating. Morphologically, neuronal calcium sensor-1 deletion leads to increased dendritic arbour complexity in the frontal cortex. At the level of hippocampal synaptic plasticity, neuronal calcium sensor-1 deletion leads to a reduction in long-term potentiation in the dentate gyrus, but not area Cornu Ammonis 1. Metabotropic glutamate receptor-induced long-term depression was unaffected in both dentate and Cornu Ammonis 1. These studies identify roles for neuronal calcium sensor-1 in specific subregions of the brain including a phenotype relevant to neuropsychiatric disorders.

International audience; Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca2+ signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca2+ transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca2+ exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca2+ transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases.

Neuronal calcium sensor 1 (NCS-1) is a high-affinity calcium-binding protein and its ubiquitous expression in the nervous system implies a wide range of functions. To date, it has been implicated in regulation of calcium channels in both axonal growth cones and presynaptic terminals, pre- and postsynaptic plasticity mechanisms, learning and memory behaviors, dopaminergic signaling, and axonal regeneration. This review summarizes these functions and relates them to several diseases in which NCS-1 plays a role, such as schizophrenia and bipolar disorder, X-linked mental retardation and fragile X syndrome, and spinal cord injury. Many questions remain unanswered about the role of NCS-1 in these diseases, particularly as the genetic factors that control NCS-1 expression in both normal and diseased states are still poorly understood. The review further identifies the therapeutic potential of manipulating the interaction of NCS-1 with its many targets and suggests directions for future research on the role of NCS-1 in these disorders.

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca(2+) concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca(2+)-bound state, the presence of Mg(2+) allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.

NCS1 (neuronal calcium sensor-1) is a Ca 2+ -myristoyl switch protein of the NCS protein family involved in synaptic plasticity and neurotransmission via Ca 2+ -dependent regulation of dopamine D2 receptor and associated Gprotein coupled receptor kinase (GRK)-2. Overexpression of NCS1 in synaptic terminals results in accumulation of membrane-bound protein and its redundant regulatory activity associated with neurological disorders. Here, we have demonstrated that bovine photoreceptors contain NCS1 that is capable of a partially irreversible interaction with isolated photoreceptor membranes and implicated in Ca 2+ -dependent binding and regulation of GRK1 in vitro. Using NCS1- recoverin C-terminal chimeric construct (NR), it was found that the Ca 2+ -myristoyl switch of NCS1 is affected by its Cterminal segment downstream the fourth EF-loop of the protein, which is variable within the NCS family. NR retains structural stability and sensitivity to Ca 2+ , but interacts with photoreceptor membranes with lower affinity in a Ca 2+ - dependent fully reversible manner and displays altered GRK1 modulation. These data combined with fluorescent probing of surface hydrophobicity of NCS1, NR and recoverin suggest that the C-terminal segment of NCS1 regulates reuptake of myristoyl group under Ca 2+ -free conditions and participates in organization of the target-binding pocket of the protein. We point out a putative role of NCS1 in photoreceptors as a modulator of GRK activity and propose targeting of the Cterminal segment of NCS1 as an appropriate way for selective suppression of excessive membrane accumulation and aberrant activity of the protein in neurons associated with central nervous system dysfunctions.

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kβ). Of a particular note, Ca(2+) induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca(2+)-binding to EF-1 is restored. Moreover, Ca(2+), which otherwise augments the enzymatic activity of PI4Kβ (modulated by NCS-1), leads to a further decline in the modulated PI4Kβ activity by myristoylated mutants (with canonical EF-1) pointing toward a loss of Ca(2+) signaling and specificity at the structural as well as functional levels. This study establishes the presence of the strong liaison between myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of Ca(2+) specific signal transduction to downstream effecter molecules despite Ca(2+) binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca(2+) sensitivity/specificity in NCS-1.

Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional Ca2+-binding protein that affects a range of cellular processes beyond those related to neurons. Functional characterization of NCS-1 in neuronal model systems suggests that NCS-1 may influence oncogenic processes. To this end, the biological role of NCS-1 was investigated by altering its endogenous expression in MCF-7 and MB-231 breast cancer cells. Overexpression of NCS-1 resulted in a more aggressive tumor phenotype demonstrated by a marked increase in invasion and motility, and a decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitaxel-induced cell death in a manner that was independent of cellular proliferation. To determine the association between NCS-1 and clinical outcome, NCS-1 expression was measured in two independent breast cancer cohorts by the Automated Quantitative Analysis method of quantitative immunofluorescence. Elevated levels of NCS-1 were significantly correlated with shorter survival rates. Furthermore, multivariate analysis demonstrated that NCS-1 status was prognostic, independent of estrogen receptor, progesterone receptor, HER2, and lymph node status. These findings indicate that NCS-1 plays a role in the aggressive behavior of a subset of breast cancers and has therapeutic or biomarker potential. Implications: NCS-1, a calcium-binding protein, is associated with clinicopathologic features of aggressiveness in breast cancer cells and worse outcome in two breast cancer patient cohorts. Mol Cancer Res; 15(7); 942–52. ©2017 AACR.

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure- function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-Terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

pH is highly regulated in the mammalian central nervous systems. Neuronal calcium sensor-1 (NCS-1) can interact with numerous target proteins. Compared with C. elegans, evolution has avoided histidine residues at positions 102 and 83 in the human and Xenopus laevis NCS-1 protein, possibly to decrease conformational sensitivity to pH gradients in synaptic processes. We used all-atom molecular dynamics (MD) simulations to investigate the effects of amino acid substitutions between species on human NCS-1 by substituting Arg102 and Ser83 for histidine (R102H and S83H) at neutral and acidic pH. Our cumulative 5 μs simulations revealed that the R102H mutation slightly increases the structural flexibility of loop L2 and the R102H(protonated) mutation decrease protein stability. Community network analysis illustrates that the R102H and S83H mutations weaken the inter-domain and strengthen the intra-domain correlations. Secondary structure contents in the S83H and the S83H(protonated) mutants are similar to those in the wild type (WT), whereas the global structural stabilities and salt-bridge probabilities decrease. This study highlights conformational dynamics effects of R102H and S83H mutations respectively on NCS-1’s local structural flexibility and global stability, while the protonated histidine decreases the stability. Thus, histidines at positions 102 and 83 may not be compatible with NCS-1’s function whether in neutral or protonated states.

Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/- ) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+ /calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production.

Neuronal calcium sensor-1 (NCS-1) protein has a variety of different neuronal functions and interacts with multiple binding partners mostly through a large solvent-exposed hydrophobic crevice (HC). A single R102Q mutation in human NCS-1 protein was demonstrated to be associated with autism disease. Solution NMR study reported that this R102Q mutant had long-range chemical shift effects on the HC and the C-terminal tail (L3). To understand the influence of the R102Q mutation on the HC and L3 of NCS-1, we have investigated the conformational dynamics and the structural flexibility of wild type (WT) NCS-1 and its R102Q mutant by conducting extensive all-atom molecular dynamics (MD) simulations. On the basis of six independent 450 ns MD simulations, we have found that the R102Q mutation in NCS-1 protein (1) dramatically reduces the flexibility of loops L2 and L3, (2) facilitates L3 in a more extended state to occupy the hydrophobic crevice to a larger extent, (3) significantly affects the intersegment salt bridges, and (4) changes the subspace of the free energy landscape of NCS-1 protein. Analysis of the salt bridge network in both WT and the R102Q variant demonstrates that the R102Q-mutation-induced salt bridge alternations play a critical role on the reduced flexibility of L2 and L3. These results reveal the important role of salt bridges on the structural properties of NCS-1 protein and that R102Q mutation disables the dynamic relocation of C-terminus, which may block the binding of NCS-1 protein to its receptors. This study may provide structural insights into the autistic spectrum disorder associated with R102Q mutation.

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.

The calcium (Ca2+) signalling system in the filamentous fungus Neurospora crassa is unique and significantly different from that in plants and animals, mainly with regard to the second messenger systems involved in Ca2+-release from internal stores (Galagan et al. 2003). The complex Ca2+ signalling system in N. crassa contains 48 Ca2+ signalling proteins including a Ca2+ and/or CaM binding protein called neuronal calcium sensor-1 (NCS-1), a Ca2+-permeable channel MID-1 and a PMCA-type Ca2+-ATPase NCA-2 (Zelter et al. 2004; Tamuli et al. 2013). Here, we show that ncs-1, mid-1 and nca-2 interactions affect growth, carotenoid accumulation, Ca2+ stress tolerance, ultraviolet (UV) survival and circadian-regulated conidiation in N. crassa. The N. crassa homologue of NCS-1 has a role in growth, Ca2+ stress tolerance and UV survival (Deka et al. 2011; Tamuli et al. 2011). MID-1 is necessary for Ca2+homeostasis in N. crassa (Lew et al. 2008). Mid-1 in Gibberella zeae has a role in growth, development and ascospore discharge (Cavinder et al. 2011). NCA-2 is responsible for adaptation to stress conditions and functions to pump Ca2+ out of the cell in N. crassa (Benito et al. 2000; Bowman et al. 2011). In Schizosaccharomyces pombe, Ncs1p, the homologue of NCS-1, promotes Ca2+-induced closure of Yam8p Ca2+ channel, a homologue of the MID1 (Hamasaki-Katagiri and Ames 2010). The Ca2+-sensitive phenotype of the ncs1 deletion mutant is rescued by a yam8 deletion, indicating that Ncs1p negatively regulates Yam8p in S. pombe

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

Neuronal calcium sensor-1 (NCS-1) protein has orthologues from Saccharomyces cerevisiae (Frq1) to human with highly conserved amino acid sequences. NCS-1 is an important factor controlling the animal’s response to temperature change. This leads us to investigate the temperature effects on the conformational dynamics of the human NCS-1 at 310 K and 316 K by extensive all-atom explicit-solvent molecular dynamics (MD) simulations and dynamic community network analysis. Four independent 500-ns MD simulations shows that the secondary structure content at 316 K is similar to that at 310 K, whereas the global protein structure is expanded at the elevated temperature. The elevated temperature results in loop 3 (L3) adopting an extended state that occupies the hydrophobic crevice, thereby blocking the binding of NCS-1 protein to its receptors. The number of suboptimal communication paths starting at residue D176 and ending at V190 in the L3 C-terminal tail is reduced with the elevation of temperature. The dynamic community network analysis suggests that the correlation between the N- and C-domain is weakened and the intradomain coupling is strengthened at 316 K. The elevation of temperature reduces the number of the salt bridges, especially in the C-domain, which may induce the conformational dynamic changes. This study suggests that the elevated temperature affects the conformational dynamics of human NCS-1 protein. Comparison of the structural dynamics of R102Q mutant and Δ176–190 truncated NCS-1 suggests that the structural and dynamics response of NCS-1 protein to elevation of temperature may be one of its intrinsic functional properties.

Peripheral neuropathy is one of the most severe and irreversible side effects caused by treatment from several chemotherapeutic drugs, including paclitaxel (Taxol®) and vincristine. Strategies are needed that inhibit this unwanted side effect without altering the chemotherapeutic action of these drugs. We previously identified two proteins in the cellular pathway that lead to Taxol-induced peripheral neuropathy, neuronal calcium sensor-1 (NCS-1) and calpain. Prolonged treatment with Taxol induces activation of calpain, degradation of NCS-1, and loss of intracellular calcium signaling. This paper has focused on understanding the molecular basis for prevention of peripheral neuropathy by testing the effects of addition of two candidate compounds to the existing chemotherapeutic drug regime: lithium and ibudilast. We found that the co-administration of either lithium or ibudilast to neuroblastoma cells that were treated with Taxol or vincristine inhibited activation of calpain and the reductions in NCS-1 levels and calcium signaling associated with these chemotherapeutic drugs. The ability of Taxol to alter microtubule formation was unchanged by the addition of either candidate compound. These results allow us to suggest that it is possible to prevent the unnecessary and irreversible damage caused by chemotherapeutic drugs while still maintaining therapeutic efficacy. Specifically, the addition of either lithium or ibudilast to existing chemotherapy treatment protocols has the potential to prevent chemotherapy-induced peripheral neuropathy.

Neuronal calcium sensor proteins and their homologues participate in transducing extracellular signals that affect intracellular Ca(2+) levels, which in turn regulate enzyme activities, secretion, gene expression and other biological processes. The filamentous fungus Beauveria bassiana is a broad-host-range pathogen of insects that acidifies the extracellular milieu during growth and pathogenesis towards target hosts. A collection of B. bassiana random insertion mutants were screened on pH indicator plates and one mutant was isolated that displayed reduced acidification. The random insertion site was mapped to a gene that displayed homology to the neuronal calcium sensor/frequenin protein family and was designated Bbcsa1. To validate the role of Bbcsa1 in B. bassiana, a targeted gene-knockout was constructed. Data confirmed that Bbcsa1 was not an essential gene and the ΔBbcsa1 strain displayed delayed acidification of the medium when grown in Czapek-Dox medium, as compared with the wild-type parent. HPLC profiling of secreted metabolites did not detect any major changes in the production of organic acids, although downregulation of the membrane H(+) pump/ATPase was noted in the mutant. A slight growth-deficient phenotype was observed for the ΔBbcsa1 strain on Czapek-Dox and potato dextrose media, which was accentuated at high calcium concentrations (500 mM) and 1.5 M sorbitol, but was unaffected by EDTA or SDS. Perturbations in vacuole morphology were also noted for the mutant. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the ΔBbcsa1 mutant when applied topically, representing the natural route of infection, but no significant effect was observed when fungal cells were directly injected into target hosts. These results suggest that Bbcsa1 participates in pre-penetration or early penetration events, but is dispensable once the insect cuticle has been breached.

Neuronal calcium sensor 1 (NCS-1) and orthologs are expressed in all organisms from yeast to humans. In the latter, NCS-1 plays an important role in neurotransmitter release and interacts with a plethora of binding partners mostly through a large solvent-exposed hydrophobic crevice. The structural basis behind the multispecific binding profile is not understood. To begin to address this, we applied NMR spectroscopy to determine the solution structure of calcium-bound human NCS-1. The structure in solution demonstrates interdomain flexibility and, in the absence of a binding partner, the C-terminal tail residues occupy the hydrophobic crevice as a ligand mimic. A variant with a C-terminal tail deletion shows lack of a defined structure but maintained cooperative unfolding and dramatically reduced global stability. The results suggest that the C-terminal tail is important for regulating the conformational stability of the Ca 2+ -activated state. Furthermore, a single amino acid mutation that was recently diagnosed in a patient with autistic spectrum disorder was seen to affect the C-terminal tail and binding crevice in NCS-1.

Calcium (Ca(2+)) is an important intracellular messenger, regulating myocyte contraction via excitation-contraction (EC) coupling and gene transcription underlying hypertrophy in the heart. Although the mechanisms of EC coupling in the immature heart are believed to be different from those in the adult heart because of the structural immaturity of the sarcoplasmic reticulum in the young heart, the details of these mechanisms are not completely understood. Neuronal Ca(2+) sensor-1 (NCS-1) is an EF-hand Ca(2+)-binding protein that is highly expressed in young hearts; however, little is known about its cardiac functions. In this review, we summarize our recent findings indicating that NCS-1 acts as a novel regulator enhancing Ca(2+) signals in the heart and hence promoting contraction in the immature heart and hypertrophy in the adult heart. Possible signal transduction pathways are also discussed.

Paclitaxel (Taxol) is one of the most effective treatment options for patients suffering from a variety of cancers. A major side effect seen in a high percentage of patients treated with paclitaxel is irreversible peripheral neuropathy. We previously reported that prolonged treatment with paclitaxel activates a calcium-dependent enzyme, calpain, which degrades neuronal calcium sensor 1 (NCS-1) and subsequent loss of intracellular calcium signaling. Because it appears that activation of calpain is an early step in this destructive cascade, we proposed that inhibition of calpain will protect against the unwanted side effects of paclitaxel treatment. First, NCS-1 levels and intracellular calcium signaling were found to be protected by the presence of lactacystin, a protesome inhibitor. To reinforce the role of calpain in this process, we showed that increased concentrations of calpastatin, a naturally occurring calpain inhibitor, were protective. Next, we tested two mutated versions of NCS-1 developed with point mutations at the P2 position of the calpain cleavage site of NCS-1 to decrease the likelihood of NCS-1 degradation. One mutant was cleaved more favorably by calpain compared with NCS-1 WT, whereas the other mutant was less favorably cleaved. Expression of either mutated version of NCS-1 in neuroblastoma cells protected intracellular calcium signals from paclitaxel-induced changes. These results support our hypothesis that it is possible to protect cells from paclitaxel-induced degradation of NCS-1 by inhibiting calpain activity.

NCU04379 gene encodes a conserved Ca(2+) and/or calmodulin binding protein that possesses a consensus signal for N-terminal myristoylation and four EF-hands, characteristics of Neuronal Calcium Sensor-1proteins. The NCU04379.2 knockout mutant shows slow growth rate, increased sensitivity to calcium and ultraviolet (UV) irradiation, and a wild-type fragment carrying NCU04379 gene complements the mutant. Therefore, NCU04379 gene has a role in growth, calcium stress tolerance, and UV survival. Crosses homozygous for ΔNCU04379.2 mutant strains were fully fertile; however, we found evidence for involvement of Ca(2+)/calmodulin-dependent protein kinase encoding genes NCU02283 and NCU09123 in sexual development.

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca2+-binding sites, and mediates the effects of cytosolic Ca2+. Minute changes in free intracellular Ca2+ are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca2+/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca2+/CaMKs comes mostly from studies of the mammalian enzymes. Thus far very few fungal homologues of NCS-1 and Ca2+/CaMKs have been characterized and little is known about their cellular roles. In this minireview, we describe the known sequences, interactions with target proteins and cellular roles of NCS-1 and Ca2+/CaMKs in fungi. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Calcium (Ca(2+)) is an important intracellular messenger underlying cell physiology. Ca(2+) channels are the main entry route for Ca(2+) into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1 (NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca(2+) sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca(2+)-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca(2+) affinity of 0.3 μM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca(2+)-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca(2+) channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1 as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIβ. NCS-1 also affects neuronal outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with Ca(2+) channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions from chromaffin cells, PC12 cells and other models, to describe how NCS-1's regulation of Ca(2+) channels allows it to exert its physiological effects.

Paclitaxel (Taxol) is a microtubule-stabilizing compound that is used for cancer chemotherapy. However, Taxol administration is limited by serious side effects including cardiac arrhythmia, which cannot be explained by its microtubule-stabilizing effect. Recently, neuronal calcium sensor 1 (NCS-1), a calcium binding protein that modulates the inositol-1,4,5-trisphosphate receptor (InsP(3)R), was described as a binding partner of Taxol and as a substrate of calpain. We examined calcium signaling processes in cardiomyocytes after treatment with Taxol to investigate the basis of Taxol-induced cardiac arrhythmia. After treating isolated neonatal rat ventricular myocytes with a therapeutic concentration of Taxol for several hours live cell imaging experiments showed that the frequency of spontaneous calcium oscillations significantly increased. This effect was not mimicked by other tubulin-stabilizing agents. However, it was prevented by inhibiting the InsP(3)R. Taxol treated cells had increased expression of NCS-1, an effect also detectable after Taxol administration in vivo. Short hairpin RNA mediated knockdown of NCS-1 decreased InsP(3)R dependent intracellular calcium release, whereas Taxol treatment, that increased NCS-1 levels, increased InsP(3)R dependent calcium release. The effects of Taxol were ryanodine receptor independent. At the single channel level Taxol and NCS-1 mediated an increase in InsP(3)R activity. Calpain activity was not affected by Taxol in cardiomyocytes suggesting a calpain independent signaling pathway. In short, our study shows that Taxol impacts calcium signaling and calcium oscillations in cardiomyocytes through NCS-1 and the InsP(3)R.

Neuronal Calcium Sensor-1 (NCS-1) is a member of calcium sensor family. It is originally identified as frequenin. NCS-1 has been found to interact with membrane and cytosolic proteins and its physiological role is governed by N-terminal myristoylation. In this paper, we report the NMR assignments of both myristoylated and non-myristoylated NCS-1 in the presence of a membrane.

Neuronal calcium sensor (NCS) proteins regulate signal transduction and are highly conserved from yeast to humans. NCS homolog in fission yeast (Ncs1p) is essential for cell growth under extreme Ca(2+) conditions. Ncs1p expression increases approximately 100-fold when fission yeast grows in high extracellular Ca(2+) (0.1 M). Here, we show that Ca(2+)-induced expression of Ncs1p is controlled at the level of transcription. Transcriptional reporter assays show that ncs1 promoter activity increased 30-fold when extracellular Ca(2+) was raised to 0.1 M and was highly Ca(2+)-specific. Ca(2+)-dependent transcription of ncs1 is abolished by the calcineurin inhibitor (FK506) and by knocking out the calcineurin target, prz1. Thus, Ca(2+)-induced expression of Ncs1p is linked to the calcineurin/prz1 stress response. The Ca(2+)-responsive ncs1 promoter region consists of 130 nucleotides directly upstream from the start codon and contains tandem repeats of the sequence, 5'-caact-3', that binds to Prz1p. The Ca(2+)-sensitive ncs1Delta phenotype is rescued by a yam8 null mutation, suggesting a possible interaction between Ncs1p and the Ca(2+) channel, Yam8p. Ca(2+) uptake and Ncs1p binding to yeast membranes are both decreased in yam8Delta, suggesting Ca(2+)-induced binding of Ncs1p to Yam8p results in channel closure. We propose that Ncs1p promotes Ca(2+) tolerance in fission yeast, in part by cytosolic Ca(2+) buffering and perhaps by negatively regulating the Yam8p Ca(2+) channel.

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1). The pharmacological inhibition of InsP(3) receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP(3)R1, which may play an important role in the regulation of neurite outgrowth.