Calcium Sensor Protein Research Articles

Phosphodiesterase 5 expression in photoreceptors rescues retinal degeneration induced by deregulation of membrane guanylyl cyclase.

Mutations in retinal membrane guanylyl cyclase 1 (RetGC1) and its calcium-sensor protein (GCAP1) cause congenital dominant retinopathies by elevation of cGMP synthesis in photoreceptors in the dark. We explored counteracting the elevated cGMP synthesis causing photoreceptor degeneration using ectopic expression of a non-photoreceptor cGMP phosphodiesterase (PDE) isozyme PDE5. PDE5 primary structure was modified to direct the delivery of the recombinant PDE5 (PDE5r) to rod outer segments (ROS), by placing a C-terminal fragment derived from a cone-specific alpha-subunit of PDE6C at the C-terminus of the PDE5, which allowed PDE5r expressed under control of mouse rod opsin promoter to accumulate in ROS. Expression of PDE5r did not affect calcium-sensitivity of RetGC regulation in PDE5rTg transgenic retinas, but increased cGMP hydrolysis in the dark, which partially desensitized PDR5rTg rods in the dark via an 'equivalent light' effect, analogous to exposure to a constant dim light of ∼20-40 photons μm-2 sec-1. The calcium-sensitivity of RetGC regulation remained drastically shifted outside the normal physiological range in hybrid R838STgPDE5rTg rods expressing both PDE5r and R838S RetGC1, the mutant causing GUCY2D dominant retinopathy, but the hybrid rods demonstrated a dramatic rescue from degeneration caused by the R838S RetGC1. In a similar fashion, PDE5r expression rescued degeneration of rods harboring Y99C GCAP1, one of the GCAP1 mutants most frequently causing GUCA1A dominant retinopathy. Our results open a possibility that ectopic expression of PDE5 can be used as an approach to rescue presently incurable dominant GUCY2D and GUCA1A retinopathies at the expense of a moderate reduction in rod light-sensitivity.

Placental extracellular vesicles from women with severe preeclampsia alter calcium homeostasis in cardiomyocytes: an ex vivo study.

Women with severe preeclampsia (sPE) exhibit a heightened risk of postpartum cardiovascular disease compared with those with normotensive pregnancies (NTP). Although placental extracellular vesicles (EVs) play a crucial role in feto-maternal communication, their impact on cardiomyocytes, particularly in the context of sPE, remains unclear. This study investigated the effect of sPE-associated placental EVs (sPE-Plex EVs) on cardiomyocyte calcium dynamics. We hypothesized that sPE-Plex EV mediates cardiomyocyte dysfunction by disrupting calcium signaling. EVs were isolated from plasma and placental explant culture (Plex) using precipitation methods and confirmed as Plex EVs by placental alkaline phosphatase (PLAP) activity and electron microscopy. Moreover, confocal microscopy confirmed the uptake of plasma EVs in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and Plex EVs by human AC-16 cardiomyocyte (hAC-16CM) cells. hiPSC-CM cells treated with sPE-EVs and hAC-16CM cells treated with sPE-Plex EVs exhibited significantly lower levels of stromal interaction molecule 1 (STIM1) and phospholamban (PLN) proteins compared with those treated with normotensive controls EVs, as confirmed by Western blot analysis. Treatment with sPE-Plex EVs also resulted in the downregulation of STIM1 and PLN proteins in murine cardiomyocyte (mCM) cells compared with treatment with NTP-Plex EVs. Our findings suggest that both plasma EVs and Plex EVs from sPE may alter calcium signaling in cardiac cells by downregulating calcium sensor proteins (STIM1 and PLN). Therefore, plasma EVs and Plex EVs from sPE pregnancies have adverse effects by altering calcium dynamics in hiPSC-CM, hAC-16CM, and mCM compared with normotensive control and potential impairment of cardiomyocyte function ex vivo.NEW & NOTEWORTHY This study unveils a novel link between the placenta and PE-linked heart dysfunction. We isolated and characterized placental EVs from pregnancies with sPE and normotensive controls. These plasma sPE-EVs, and sPE-Plex EVs disrupt calcium signaling in heart cells, potentially via reduced STIM1 and PLN proteins. This suggests both plasma sPE-EVs and sPE-Plex EVs cargo drive these disruptive effects. Identifying these cargo molecules (miRNAs or proteins) holds promise for new PE therapies targeting cardiac dysfunction.

A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging

In vivo imaging of large-scale neuronal activity plays a pivotal role in unraveling the function of the brain's circuitry. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to optimize three-photon microscopy for large field-of-view imaging at depths unreachable by two-photon microscopy. These techniques enable us to image neuronal activities of transgenic animals expressing protein calcium sensors in a ~ 3.5-mm diameter field-of-view with single-cell resolution in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into typical multiphoton microscopes to enlarge field of view for system-level neural circuit research.

Expression interplay of genes coding for calcium-binding proteins and transcription factors during the osmotic phase provides insights on salt stress response mechanisms in bread wheat

Bread wheat is an important crop for the human diet, but the increasing soil salinization is reducing the yield. The Ca2+ signaling events at the early stages of the osmotic phase of salt stress are crucial for the acclimation response of the plants through the performance of calcium-sensing proteins, which activate or repress transcription factors (TFs) that affect the expression of downstream genes. Physiological, genetic mapping, and transcriptomics studies performed with the contrasting genotypes Syn86 (synthetic, salt-susceptible) and Zentos (elite cultivar, salt-tolerant) were integrated to gain a comprehensive understanding of the salt stress response. The MACE (Massive Analysis of cDNA 3ʹ-Ends) based transcriptome analysis until 4 h after stress exposure revealed among the salt-responsive genes, the over-representation of genes coding for calcium-binding proteins. The functional and structural diversity within this category was studied and linked with the expression levels during the osmotic phase in the contrasting genotypes. The non-EF-hand category from calcium-binding proteins was found to be enriched for the susceptibility response. On the other side, the tolerant genotype was characterized by a faster and higher up-regulation of genes coding for proteins with EF-hand domain, such as RBOHD orthologs, and TF members. This study suggests that the interplay of calcium-binding proteins, WRKY, and AP2/ERF TF families in signaling pathways at the start of the osmotic phase can affect the expression of downstream genes. The identification of SNPs in promoter sequences and 3ʹ -UTR regions provides insights into the molecular mechanisms controlling the differential expression of these genes through differential transcription factor binding affinity or altered mRNA stability.

Characterization of the Calmodulin-Like Protein Family in Chara braunii and their Conserved Interaction with the Calmodulin-Binding Transcription Activator Family.

Calcium sensor proteins play important roles by detecting changes in intracellular calcium and relaying that information onto downstream targets through protein-protein interaction. Very little is known about calcium sensors from plant species that predate land colonization and the evolution of embryophytes. Here, we examined the genome of the multicellular algae, Chara braunii, for orthologs to the evolutionarily conserved calcium sensor calmodulin (CaM) and for CaM-like (CML) proteins. We identified one CaM and eight CML isoforms that range in size from 16.4 to 21.3 kDa and are predicted to have between two to four calcium-binding (EF-hand) domains. Using recombinant protein, we tested whether CbCaM and CbCML1-CbCML7 possess biochemical properties of typical calcium sensors. CbCaM and the CbCMLs all displayed high-affinity calcium binding with estimated global KD,app values in the physiological µM range. In response to calcium binding, CbCaM and the CbCMLs exhibited varying degrees of increase in exposed hydrophobicity, suggesting that different calcium-induced conformational changes occur among isoforms. We found many examples of putative CaM targets encoded in the C. braunii genome and explored the ability of CbCaM and CbCMLs to interact in planta with a representative putative target, a C. braunii CaM-binding transcription factor (CbCAMTA1). CbCaM, CbCML2 and CbCML4 each associated with the C-terminal region of CbCAMTA1. Collectively, our data support the hypothesis that complex calcium signaling and sensing networks involving CaM and CMLs evolved early in the green lineage. Similarly, it seems likely that calcium-mediated regulation of transcription occurs in C. braunii via CAMTAs and is an ancient trait predating embryophytic emergence.

Axonal damage and inflammation response are biological correlates of decline in small-world values: a cohort study in autosomal dominant Alzheimer's disease.

The grey matter of the brain develops and declines in coordinated patterns during the lifespan. Such covariation patterns of grey matter structure can be quantified as grey matter networks, which can be measured with magnetic resonance imaging. In Alzheimer's disease, the global organization of grey matter networks becomes more random, which is captured by a decline in the small-world coefficient. Such decline in the small-world value has been robustly associated with cognitive decline across clinical stages of Alzheimer's disease. The biological mechanisms causing this decline in small-world values remain unknown. Cerebrospinal fluid (CSF) protein biomarkers are available for studying diverse pathological mechanisms in humans and can provide insight into decline. We investigated the relationships between 10 CSF proteins and small-world coefficient in mutation carriers (N = 219) and non-carriers (N = 136) of the Dominantly Inherited Alzheimer Network Observational study. Abnormalities in Amyloid beta, Tau, synaptic (Synaptosome associated protein-25, Neurogranin) and neuronal calcium-sensor protein (Visinin-like protein-1) preceded loss of small-world coefficient by several years, while increased levels in CSF markers for inflammation (Chitinase-3-like protein 1) and axonal injury (Neurofilament light) co-occurred with decreasing small-world values. This suggests that axonal loss and inflammation play a role in structural grey matter network changes.

S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and β-adrenergic receptor (βAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during βAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

Blockade of mGluR5 in nucleus accumbens modulates calcium sensor proteins, facilitates extinction, and attenuates reinstated morphine place preference in rats

Numerous findings confirm that the metabotropic glutamate receptors (mGluRs) are involved in the conditioned place preference (CPP) induced by morphine. Here we focused on the role of mGluR5 in the nucleus accumbens (NAc) as a main site of glutamate action on the rewarding effects of morphine. Firstly, we investigated the effects of intra-NAc administrating mGluR5 antagonist 3-((2-Methyl-1,3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP; 1, 3, and 10 μg/μl saline) on the extinction and the reinstatement phase of morphine CPP. Moreover, to determine the downstream signaling cascades of mGluR5 in morphine CPP, the protein levels of stromal interaction molecules (STIM1 and 2) in the NAc and hippocampus (HPC) were measured by western blotting. The behavioral data indicated that the mGluR5 blockade by MTEP at the high doses of 3 and 10 μg facilitated the extinction of morphine-induced CPP and attenuated the reinstatement to morphine in extinguished rats. Molecular results showed that the morphine led to increased levels of STIM proteins in the HPC and increased the level of STIM1 without affecting STIM2 in the NAc. Furthermore, intra-NAc microinjection of MTEP (10 μg) in the reinstatement phase decreased STIM1 in the NAc and HPC and reduced the STIM2 in the HPC. Collectively, our data show that morphine could facilitate brain reward function in part by increasing glutamate-mediated transmission through activation of mGluR5 and modulation of STIM proteins. Therefore, these results highlight the therapeutic potential of mGluR5 antagonists in morphine use disorder.

Pharmacoinformational, gerontoinformational and chemoreactomic analysis of the molecule of citrulline malate, carnitine, sulbutiamine and meldonium to identify the molecular mechanisms of antiasthenic action

The pathophysiology of asthenia is very complex and is associated with chronic kidney disease, heart failure, chronic obstructive pulmonary disease, sarcopenia, bacterial and viral pathogens, micronutrient nutritional imbalances, hypothyroidism, etc. Asthenia can occur with excessive (for a given patient) physical, mental or mental stress and adaptation disorders or be iatrogenic in nature (in particular, due to taking medications that contribute to increased loss of vitamins and microelements), incl. due to unwanted drug interactions. The complex nature of the pathophysiology of asthenia necessitates the use of a differentiated approach aimed at eliminating the main cause of asthenia in a given patient. If asthenia is associated primarily with disorders of energy metabolism, then the pathophysiological treatment is the use of nutrients that support intracellular synthesis - such as citrulline, citrulline malate, the main mechanisms of action of which are supporting the urea cycle, increasing the excretion of ammonium ions, reducing the concentration lactate in the blood. The paper presents the results of a comparative pharmacoinformatic and chemoreactomic analysis of citrulline, citrulline malate (CM), carnitine, sulbutiamine and meldonium. The profile of pharmacological effects of citrulline/CM was significantly different from the profiles of other molecules. For citrulline/CM, cholinergic, antidepressant, and lipid-modifying effects have been identified and an antiasthenic effect has been suggested when used in the treatment of Duchenne muscular dystrophy and for disorders of carbohydrate metabolism. Unlike other molecules, CM and carnitine do not contribute to the loss of vitamins and minerals. Inhibition of the CM serotonin 5HT3A receptor may improve vestibulation because blockers of 5-HT3 receptors concentrated in neurons of the vestibular apparatus, improves tests of balance and walking in an experiment in mice. A positive dose-dependent effect of citrulline and CM on the lifespan of a number of model organisms has been shown. Chemoreactomic analysis of molecular receptor proteins indicated new molecular mechanisms of the antiasthenic action of CM: inhibition of serotonin receptors, calcium sensor protein receptors, chemokine receptors, lipopolysaccharides (toll receptors), nociceptin, glutamate, orexin, purines and prostanoids, biosynthesis of NF-kB and TNFα.

Molecular recording of calcium signals via calcium-dependent proximity labeling.

Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, calcium transients play a central role in regulating neurotransmitter release and synaptic plasticity. However, it is challenging to capture the molecular events associated with these localized and ephemeral calcium signals. Here we present an engineered biotin ligase that generates permanent molecular traces in a calcium-dependent manner. The enzyme, calcium-dependent BioID (Cal-ID), biotinylates nearby proteins within minutes in response to elevated local calcium levels. The biotinylated proteins can be identified via mass spectrometry and visualized using microscopy. In neurons, Cal-ID labeling is triggered by neuronal activity, leading to prominent protein biotinylation that enables transcription-independent activity labeling in the brain. In summary, Cal-ID produces a biochemical record of calcium signals and neuronal activity with high spatial resolution and molecular specificity.

Feedforward and feedback mechanisms cooperatively regulate rapid experience-dependent response adaptation in a single thermosensory neuron type

Sensory adaptation allows neurons to adjust their sensitivity and responses based on recent experience. The mechanisms that mediate continuous adaptation to stimulus history over seconds- to hours-long timescales, and whether these mechanisms can operate within a single sensory neuron type, are unclear. The single pair of AFD thermosensory neurons in Caenorhabditis elegans exhibits experience-dependent plasticity in their temperature response thresholds on both minutes- and hours-long timescales upon a temperature upshift. While long-term response adaptation requires changes in gene expression in AFD, the mechanisms driving rapid response plasticity are unknown. Here, we show that rapid thermosensory response adaptation in AFD is mediated via cGMP and calcium-dependent feedforward and feedback mechanisms operating at the level of primary thermotransduction. We find that either of two thermosensor receptor guanylyl cyclases (rGCs) alone is sufficient to drive rapid adaptation, but that each rGC drives adaptation at different rates. rGC-driven adaptation is mediated in part via phosphorylation of their intracellular domains, and calcium-dependent feedback regulation of basal cGMP levels via a neuronal calcium sensor protein. In turn, cGMP levels feedforward via cGMP-dependent protein kinases to phosphorylate a specific subunit of the cGMP-gated thermotransduction channel to further regulate rapid adaptation. Our results identify multiple molecular pathways that act in AFD to ensure rapid adaptation to a temperature change and indicate that the deployment of both transcriptional and nontranscriptional mechanisms within a single sensory neuron type can contribute to continuous sensory adaptation.

Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Copines, a Family of Calcium Sensor Proteins and Their Role in Brain Function.

The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share a unique architecture and contain tandem C2 domains and a Von Willebrand factor type A (VWA) domain. C2 domains in Copines bind calcium, phospholipids, and other proteins and mediate the transient association of these proteins with biological membranes at elevated calcium levels. The VWA domain also binds calcium and is involved in protein-protein interactions. Here, we provide a comprehensive review of the sequences, structures, expression, targeting, and function of the entire family of known Copine proteins (Copine 1-9 in mammals) with a particular emphasis on their functional roles in the mammalian brain. Neuronal Copines are implicated in a wide array of processes from cell differentiation to synaptic transmission and plasticity and are also linked to several pathological conditions from cancers to brain diseases. This review provides the most up-to-date insights into the structure and function of Copines, with an emphasis on their role in brain function.

Generation of an NCS1 gene knockout human induced pluripotent stem cell line using CRISPR/Cas9

NCS1 (Neuronal calcium sensor protein 1) encodes a highly conserved calcium binding protein abundantly expressed in neurons. It modulates intracellular calcium homeostasis, calcium-dependent signaling pathways as well as neuronal transmission and plasticity. Here, we generated a NCS1 knockout human induced pluripotent stem cell (hiPSC) line using CRISPR-Cas9 genome editing. It shows regular expression of pluripotent markers, normal iPSC morphology and karyotype as well as no detectable off-target effects on top 6 potentially affected genes. This newly generated cell line constitutes a valuable tool for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological disease.

Involvement of the Calmodulin-like Protein Gene VaCML92 in Grapevine Abiotic Stress Response and Stilbene Production.

Calmodulin-like proteins (CMLs) are an important family of plant calcium sensor proteins that sense and decode changes in the intracellular calcium concentration in response to environmental and developmental stimuli. Nonetheless, the specific functions of individual CML family members remain largely unknown. This study aims to explore the role of the Vitis amurensis VaCML92 gene in the development of its high stress resistance and the production of stilbenes. The expression of VaCML92 was sharply induced in V. amurensis cuttings after cold stress. The VaCML92 gene was cloned and its role in the abiotic stress responses and stilbene production in grapevine was further investigated. The VaCML92-overexpressing callus cell cultures of V. amurensis and soil-grown plants of Arabidopsis thaliana exhibited enhanced tolerance to cold stress and, to a lesser extent, to the drought, while their tolerance to heat stress and high salinity was not affected. In addition, the overexpression of VaCML92 increased stilbene production in the V. amurensis cell cultures by 7.8-8.7-fold. Taken together, the data indicate that the VaCML92 gene is involved as a strong positive regulator in the rapid response to cold stress, the induction of cold stress resistance and in stilbene production in wild grapevine.

Visualizing the activation of encephalitogenic T cells in the ileal lamina propria by in vivo two-photon imaging

Autoreactive encephalitogenic T cells exist in the healthy immune repertoire but need a trigger to induce CNS inflammation. The underlying mechanisms remain elusive, whereby microbiota were shown to be involved in the manifestation of CNS autoimmunity. Here, we used intravital imaging to explore how microbiota affect the T cells as trigger of CNS inflammation. Encephalitogenic CD4+ T cells transduced with the calcium-sensing protein Twitch-2B showed calcium signaling with higher frequency than polyclonal T cells in the small intestinal lamina propria (LP) but not in Peyer's patches. Interestingly, nonencephalitogenic T cells specific for OVA and LCMV also showed calcium signaling in the LP, indicating a general stimulating effect of microbiota. The observed calcium signaling was microbiota and MHC class II dependent as it was significantly reduced in germfree animals and after administration of anti-MHC class II antibody, respectively. As a consequence of T cell stimulation in the small intestine, the encephalitogenic T cells start expressing Th17-axis genes. Finally, we show the migration of CD4+ T cells from the small intestine into the CNS. In summary, our direct in vivo visualization revealed that microbiota induced T cell activation in the LP, which directed T cells to adopt a Th17-like phenotype as a trigger of CNS inflammation.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

Expansion Microscopy Application for Calcium Protein Clustering Imaging in Cells and Brain Tissues.

Many biological studies require high-resolution imaging and subsequent analysis of cell organelles and molecules. Some membrane proteins form tight clusters, and this process is directly linked to their function. In most studies, these small protein clusters have been investigated by total internal reflection fluorescence (TIRF) microscopy, which enables imaging with high spatial resolution within 100 nm of the membrane surface. Recently developed expansion microscopy (ExM) makes it possible to achieve nanometer resolution using a conventional fluorescence microscope by physically expanding the sample. In this article, we describe implementation of ExM for imaging of protein clusters formed by the endoplasmic reticulum (ER) calcium sensor protein STIM1. This protein translocates during ER store depletion and forms clusters that support contact with plasma membrane (PM) calcium-channel proteins. ER calcium channels such as the type 1 inositol triphosphate receptor (IP3R) also form clusters, but their investigation by TIRF microscopy is impossible due to the large distance from the PM. In this article, we demonstrate how to investigate IP3R clustering using ExM in hippocampal brain tissues. We compare IP3R clustering in the CA1 area of the hippocampus of wild-type and 5xFAD Alzheimer's disease model mice. To facilitate future applications, we describe experimental protocols and image processing guidelines for application of ExM to membrane and ER protein clustering studies in cultured cells and brain tissues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Expansion microscopy application for protein cluster visualization in cells Alternate Protocol: Expansion microscopy application for protein cluster visualization in brain tissues Basic Protocol 2: Protein cluster analysis of expansion microscopy images using ImageJ and Icy software.

Molecular tuning of calcium dependent processes by neuronal calcium sensor proteins in the retina

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca2+. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca2+-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca2+-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca2+-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca2+-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.