Search Results (216)

Calmodulins (CaMs) and calmodulin-like proteins (CMLs) belong to families of calcium-sensors that act as calcium ion (Ca²⁺) signal-decoding proteins and regulate downstream target proteins. As a tropical halophyte, Canavalia rosea shows great resistance to multiple abiotic stresses, including high salinity/alkalinity, extreme drought, heat, and intense sunlight. However, investigations of calcium ion signal transduction involved in the stress responses of C. rosea are limited. The CaM and CML gene families have been identified and characterized in many other plant species. Nevertheless, there is limited available information about these genes in C. rosea. In this study, a bioinformatic analysis, including the gene structures, conserved protein domains, phylogenetic relationships, chromosome distribution, and gene synteny, was comprehensively performed to identify and characterize CrCaMs and CrCMLs. A spatio-temporal expression assay in different organs and environmental conditions was then conducted using the RNA sequencing technique. Additionally, several CrCaM and CrCML members were then cloned and functionally characterized using the yeast heterogeneous expression system, and some of them were found to change the tolerance of yeast to heat, salt, alkalinity, and high osmotic stresses. The results of this study provide a foundation for understanding the possible roles of the CrCaM and CrCML genes, especially for halophyte C. rosea’s natural ecological adaptability for its native habitats. This study also provides a theoretical basis for further study of the physiological and biochemical functions of plant CaMs and CMLs that are involved in tolerance to multiple abiotic stresses. Full article

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K⁺ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (I_K1) and the rapid delayed rectifier (I_Kr) K⁺ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. I_K1 and I_Kr were dissected by 50 µM BaCl₂ and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased I_K1 under the plateau phase of the action potential (0–+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall I_K1 integral. The ISO-induced enhancement of I_K1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the I_K1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the I_Kr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances I_K1 in canine left ventricular cells through the CaMKII pathway, while I_Kr remains unaffected under the experimental conditions used. Full article

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII. Full article

Since even low-level environmental exposure to cadmium (Cd) can lead to numerous unfavourable health outcomes, including damage to the nervous system, it is important to recognize the risk of health damage by this xenobiotic, the mechanisms of its toxic influence, and to find an effective protective strategy. This study aimed to evaluate, in a female Wistar rat model of current human environmental exposure to Cd (1 and 5 mg/kg of diet for 3–24 months), if the low-to-moderate treatment with this element can harm the brain and whether the supplementation with a 0.1% Aronia melanocarpa L. (Michx.) Elliott berries (chokeberries) extract (AE) can protect against this effect. The exposure to Cd modified the values of various biomarkers of neurotoxicity, including enzymes (acetylcholinesterase (AChE), sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase), phospholipase A2 (PLA2), and nitric oxide synthase 1 (NOS1)) and non-enzymatic proteins (calmodulin (CAM), nuclear factor erythroid 2-related factor 2 (Nrf2), and Kelch-like ECH-associated protein 1 (KEAP1)) crucial for the functioning of the nervous system, as well as the concentrations of calcium (Ca) and magnesium (Mg) and some metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the brain tissue. The co-administration of AE, partially or entirely, protected from most of the Cd-induced changes alleviating its neurotoxic influence. In conclusion, even low-level chronic exposure to Cd may adversely affect the nervous system, whereas the supplementation with A. melanocarpa berries products during the treatment seems a protective strategy. Full article

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca²⁺_i) at concentrations ([Ca²⁺]_i) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca²⁺-binding sites, most likely gating is mediated by a Ca²⁺-binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca²⁺-CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others. Based on these data, since 2000, we have published a Ca²⁺-CaM-cork gating model. Despite convincing evidence for the Ca²⁺-CaM role in gating, a recent study has proposed an alternative gating model that would involve a direct electrostatic Ca²⁺-connexin interaction. However, this study, which tested the effect of unphysiologically high [Ca²⁺]_i on the structure of isolated junctions, reported that neither changes in the channel’s pore diameter nor connexin conformational changes are present, in spite of exposure of isolated gap junctions to [Ca²⁺]_i as high at the 20 mM. In conclusion, data generated in the past four decades by multiple experimental approaches have clearly demonstrated the direct role of Ca²⁺-CaM in gap junction channel gating. Full article

Magnolia wufengensis, a newly discovered ornamental species in the Magnoliaceae family, is susceptible to salinity. Moreover, Ca²⁺ is an essential element for plant growth and is receiving increasing attention for its ability to mitigate the negative effects of environmental stress on plants. In the present study, we investigated the effect of Ca²⁺ on the growth and transcriptome of M. wufengensis under salt stress. The treatments used here were as follows: control, NaCl (150 mmol/L), CaCl₂ (5 mmol/L), and NaCl (150 mmol/L) + CaCl₂ (5 mmol/L). After a 60-day treatment period, plant growth indices were determined, and leaves were collected for physiological analysis and transcriptome investigation. The combined application of NaCl and CaCl₂ alleviated phenotypic damage and restored seedling growth. Moreover, RNA sequencing data revealed that in the Na vs. control group and the NaCa vs. Na group, there were 968 and 2632 differentially expressed genes, respectively, which were both primarily enriched in secondary metabolism, glutathione metabolism, signaling hormone metabolism, glucose metabolism, and amino acid metabolism. These pathways were analyzed to screen key genes: the adenosine triphosphate (ATP)-binding cassette efflux transporter G1 (ABCG1) genes, which are related to transmembrane transport; the calmodulin genes, which are related to signal transmission; and the glutathione S-transferase (GST), glutathione peroxidase (GPX), and peroxidase (POD) genes related to antioxidant enzymes. Lastly, we constructed a hypothesis model of Ca²⁺-enhanced salt tolerance in M. wufengensis. This study reveals the potential mechanisms by which Ca²⁺ enhances the salt tolerance of M. wufengensis and provides a theoretical reference for its cultivation in saline areas. Full article

Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca²⁺/CaM)-dependent serine/threonine (Ser/Thr) protein kinase and is characteristically downregulated in metastatic cancer. Several studies showed that DAPK1 is involved in both the early and late stages of cancer. DAPK1 downregulation is elaborately controlled by epigenetic, transcriptional, posttranscriptional, and posttranslational processes. DAPK1 is known to regulate not only cancer cells but also stromal cells. Recent studies showed that DAPK1 was involved not only in tumor suppression but also in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) formation in colon and thyroid cancers. CSCs are major factors in determining cancer aggressiveness in cancer metastasis and treatment prognosis by influencing EMT. However, the molecular mechanism involved in the regulation of cancer cells by DAPK1 remains unclear. In particular, little is known about the existence of CSCs and how they are regulated in papillary thyroid carcinoma (PTC) among thyroid cancers. In this review, we describe the molecular mechanism of CSC regulation by DAPK1 in PTC progression. Full article

Clinical ketosis is a detrimental metabolic disease in dairy cows, often accompanied by severe lipolysis and inflammation in adipose tissue. Our previous study suggested a 2.401-fold upregulation in the calmodulin (CaM) level in the adipose tissue of cows with clinical ketosis. Therefore, we hypothesized that CaM may regulate lipolysis and inflammatory responses in cows with clinical ketosis. To verify the hypothesis, we conducted a thorough veterinary assessment of clinical symptoms and serum β-hydroxybutyrate (BHB) concentration. Subsequently, we collected subcutaneous adipose tissue samples from six healthy and six clinically ketotic Holstein cows at 17 ± 4 days postpartum. Commercial kits were used to test the abundance of BHB, non-esterified fatty acid (NEFA), the liver function index (LFI), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). We found that cows with clinical ketosis exhibited higher levels of BHB, NEFA, LFI, IL-6, IL-1β, TNF-α, and lower glucose levels than healthy cows. Furthermore, the abundance of CaM, toll-like receptor 4 (TLR4), inhibitor of nuclear factor κB kinase subunit β (IKK), phosphorylated nuclear factor κB p65/nuclear factor κB p65 (p-NF-κB p65/NF-κB p65), adipose triacylglycerol lipase (ATGL), and phosphorylated hormone-sensitive lipase/hormone-sensitive lipase (p-HSL/HSL) was increased, while that of perilipin-1 (PLIN1) was decreased in the adipose tissue of cows with clinical ketosis. To investigate the mechanism underlying the responses, we isolated the primary bovine adipocytes from the adipose tissue of healthy cows and induced the inflammatory response mediated by TLR4/IKK/NF-κB p65 with lipopolysaccharide (LPS). Additionally, we treated the primary bovine adipocytes with CaM overexpression adenovirus and CaM small interfering RNA. In vitro, LPS upregulated the abundance of TLR4, IKK, p-NF-κB p65, ATGL, p-HSL/HSL, and CaM and downregulated PLIN1. Furthermore, CaM silencing downregulated the abundance of LPS-activated p-HSL/HSL, TLR4, IKK, and p-NF-κB p65 and upregulated PLIN1 in bovine adipocytes, except for ATGL. However, CaM overexpression upregulated the abundance of LPS-activated p-HSL/HSL, TLR4, IKK, and p-NF-κB p65 and downregulated PLIN1 expression in bovine adipocytes. These data suggest that CaM promotes lipolysis in adipocytes through HSL and PINL1 while activating the TLR4/IKK/NF-κB inflammatory pathway to stimulate an inflammatory response. There is a positive feedback loop between CaM, lipolysis, and inflammation. Inhibiting CaM may act as an adaptive mechanism to alleviate metabolic dysregulation in adipose tissue, thereby relieving lipolysis and inflammatory responses. Full article

This study aimed to reveal the impact of MeJA and ZnSO₄ treatments on the physiological metabolism of barley seedlings and the content of phenolic acid. The results showed that MeJA (100 μM) and ZnSO₄ (4 mM) treatments effectively increased the phenolic acid content by increasing the activities of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase (PAL) and cinnamic acid 4-hydroxylase (C4H) and by up-regulating the expression of genes involved in phenolic acid synthesis. As a result of the MeJA or ZnSO₄ treatment, the phenolic acid content increased by 35.3% and 30.9% at four days and by 33.8% and 34.5% at six days, respectively, compared to the control. Furthermore, MeJA and ZnSO₄ treatments significantly increased the malondialdehyde content, causing cell membrane damage and decreasing the fresh weight and seedling length. Barley seedlings responded to MeJA- and ZnSO₄-induced stress by increasing the activities of antioxidant enzymes and controlling their gene expression levels. Meanwhile, MeJA and ZnSO₄ treatments significantly upregulated calcium-adenosine triphosphate, calmodulin-dependent protein kinase-related kinase, and calmodulin-dependent protein genes in barley seedlings. This suggested that Ca²⁺ may be the signaling molecule that promotes phenolic acid synthesis under MeJA and ZnSO₄ treatment. This study deepens the understanding of the phenolic acid enrichment process in barley seedlings under MeJA and ZnSO₄ treatments. Full article

Calmodulin (Calm), a crucial Ca²⁺ sensor, plays an important role in calcium-dependent signal transduction cascades. However, the expression and the relevance of Calm in stress and immune response have not been characterized in Megalobrama amblycephala. In this study, we identified the full-length cDNA of Calm (termed MaCalm) in blunt snout bream M. amblycephala, and analyzed MaCalm expression patterns in response to cadmium and Aeromonas hydrophila challenges. MaCalm was 1603 bp long, including a 5′-terminal untranslated region (UTR) of 97 bp, a 3′-terminal UTR of 1056 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculated molecular weight (MW) of 16.84 kDa and an isoelectric point (pI) of 4.09. Usually, MaCalm contains four conservative EF hand motifs. The phylogenetic tree analysis indicated that the nucleotide sequence of MaCalm specifically clustered with Ctenopharyngodon idella with high identity (98.33%). Tissue distribution analysis demonstrated that the ubiquitous expression of MaCalm mRNA was found in all tested tissues, with the highest expression in the brain and the lowest expression in muscle. MaCalm showed significant upregulation at 14 d and 28 d post exposure to varying concentrations of cadmium in the liver; HSP70 transcripts in the liver significantly upregulated at 14 d post exposure to different concentrations of cadmium. Moreover, in response to the A. hydrophila challenge in vivo, MaCalm transcripts in the liver first increased and then decreased, but MaCalm transcripts in the kidney declined gradually with prolonged infection. After the A. hydrophila challenge, the expression level of HSP70 was significantly downregulated at 24 h in the liver and its expression level was notably downregulated at 12 h and at 24 h in the kidney. Collectively, our results suggest that MaCalm possesses vital roles in stress and immune response in M. amblycephala. Full article

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca²⁺ levels, and of the heart rate. Full article

Ca²⁺ binding to the ubiquitous Ca²⁺ sensing protein calmodulin (CaM) activates the intermediate conductance Ca²⁺-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K⁺ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K⁺ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca²⁺-dependent gating process. Full article

Pulsed field ablation (PFA) is a promising new treatment for atrial fibrillation (AF), in which pulmonary vein isolation is achieved by irreversible electroporation. Electroporation causes ATP to leak through the permeabilized membrane. ATP is required both for the healing of the cell membrane and for the functioning of ion pumps, such as sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) or Na⁺,K⁺-ATPase (NKA), which play a key role in maintaining continuous contractions of the heart muscle. We investigated the effects of electroporation on the expression of ion pumps and possible correlations with the activation of AMPK, the main energy sensor in cells. H9c2 rat cardiac cells were exposed to either monopolar or bipolar (H-FIRE) pulses. Cells lysed 4 or 24 h after electroporation were used for mRNA and protein expression analyses. Overall, both pulse protocols caused a dose-dependent downregulation of crucial SERCA and NKA isoforms, except for NKAα2 and β3, which were upregulated after 24 h. Monopolar pulses also decreased the phosphorylation of FXYD1, which may cause an inhibition of NKA activity. Both pulse protocols caused an increased AMPK activity, which may decrease both SERCA and NKA activity via calcium/calmodulin-dependent protein kinase. Our results provide important new insights into what happens in surviving cardiomyocytes after they are exposed to PFA. Full article

Semaglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has gained considerable attention as a therapeutic agent for type 2 diabetes mellitus and obesity. Despite its clinical success, the precise mechanisms underlying its pharmacological effects remain incompletely understood. In this study, we employed ligand-based drug design strategies to investigate potential off-target interactions of semaglutide. Through a comprehensive in silico screening of semaglutide’s structural properties against a diverse panel of proteins, we have identified calmodulin (CaM) as a putative novel target of semaglutide. Molecular docking simulations revealed a strong interaction between semaglutide and CaM, characterized by favourable binding energies and a stable binding pose. Further molecular dynamics simulations confirmed the stability of the semaglutide–CaM complex, emphasizing the potential for a physiologically relevant interaction. In conclusion, our ligand-based drug design approach has uncovered calmodulin as a potential novel target of semaglutide. This discovery sheds light on the complex pharmacological profile of semaglutide and offers a promising direction for further research into the development of innovative therapeutic strategies for metabolic disorders. The CaM, and especially so the CaMKII, system is central in the experience of both drug- and natural-related reward. It is here hypothesized that, due to semaglutide binding, the reward pathway-based calmodulin system may be activated, and/or differently regulated. This may result in the positive semaglutide action on appetitive behaviour. Further studies are required to confirm these findings. Full article

Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the K_Ca1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1β production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1β production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions. Full article