Gossypium hirsutum calmodulin-like protein (CML 11) interaction with geminivirus encoded protein using bioinformatics and molecular techniques.

Plant viruses are the most abundant destructive agents that exist in every ecosystem, causing severe diseases in multiple crops worldwide. Currently, a major gap is present in computational biology determining plant viruses interaction with its host. We lay out a strategy to extract virus-host protein interactions using various protein binding and interface methods for Geminiviridae, a second largest virus family. Using this approach, transcriptional activator protein (TrAP/C2) encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMV) showed strong binding affinity with calmodulin-like (CML) protein of Gossypium hirsutum (Gh-CML11). Higher negative value for the change in Gibbs free energy between TrAP and Gh-CML11 indicated strong binding affinity. Consensus from gene ontology database and in-silico nuclear localization signal (NLS) tools identified subcellular localization of TrAP in the nucleus associated with Gh-CML11 for virus infection. Data based on interaction prediction and docking methods present evidences that full length and truncated C2 strongly binds with Gh-CML11. This computational data was further validated with molecular results collected from yeast two-hybrid, bimolecular fluorescence complementation system and pull down assay. In this work, we also show the outcomes of full length and truncated TrAP on plant machinery. This is a first extensive report to delineate a role of CML protein from cotton with begomoviruses encoded transcription activator protein.

Cheonwangbosimdan mitigates post-traumatic stress disorder-like behaviors through GluN2B-containing NMDA receptor antagonism in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cheonwangbosimdan (CWBSD), a herbal medicine traditionally used for anxiety, insomnia, depression, and heart palpitations, has been reported to have anti-anxiety, antidepressant, cognitive improvement, and neuroprotective effects. AIM OF THE STUDY: The purpose of this study was to determine if CWBSD could affect post-traumatic stress disorder (PTSD)-like behaviors because it has prioritized clinical use over mechanism study. MATERIALS AND METHODS: A single prolonged stress (SPS) mouse model, a well-established animal model of PTSD, was used to investigate whether standardized CWBSD could mitigate PTSD-like behaviors through robust behavioral tests, including the elevated plus-maze test and marble burying test for measuring anxiety-like behaviors, the splash test, forced swimming test, and tail suspension test for evaluating depression-like behaviors, and the Y-maze test and novel object recognition test for assessing cognitive function. Additionally, a fear extinction test was employed to determine whether CWBSD might reverse fear memory extinction deficits. Amygdala tissue was isolated from SPS-treated mouse brain and subjected to Western blotting or quantitative PCR to explore mechanisms by which CWBSD could mitigate PTSD-like behaviors. RESULTS: CWBSD ameliorated emotional impairments and cognitive dysfunction in an SPS-induced PTSD-like mouse model. It also mitigated deficits in abnormal fear memory extinction. Protein expression levels of N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) and phosphorylation levels of Ca2+/calmodulin-dependent protein kinase II in the amygdala were increased in SPS model mice and normalized by CWBSD. Additionally, co-administration of CWBSD and GluN2B-containing NMDA receptor antagonist, ifenprodil, at each sub-effective dose promoted fear memory extinction. CONCLUSIONS: CWBSD can alleviate SPS-induced PTSD-like behaviors by normalizing GluN2B-containing NMDA receptor activity in the amygdala. Therefore, CWBSD could be a promising candidate for PTSD treatment with fewer adverse effects and better efficacy than existing therapies.

A calmodulin-like protein PvCML9 negatively regulates salt tolerance.

Calmodulin-like proteins (CMLs) are unique Ca2+ sensors and play crucial roles in response to abiotic stress in plants. A salt-repressed PvCML9 from halophyte seashore paspalum (Paspalum vaginatum O. Swartz) was identified. PvCML9 was localized in the cytoplasm and nucleus and highly expressed in roots and stems. Overexpression of PvCML9 led to reduced salt tolerance in rice and seashore paspalum, whereas downregulating expression of PvCML9 showed increased salt tolerance in seashore paspalum as compared with the wild type (WT), indicating that PvCML9 regulated salt tolerance negatively. Na+ and K+ homeostasis was altered by PvCML9 expression. Lower level of Na+/K+ ratio in roots and shoots was maintained in PvCML9-RNAi lines compared with WT under salt stress, but higher level in overexpression lines. Moreover, higher levels of SOD and CAT activities and proline accumulation were observed in PvCML9-RNAi lines compared with WT under salt stress, but lower levels in overexpression lines, which altered ROS homeostasis. Based on the above data, mutation of its homolog gene OsCML9 in rice by CRISPR/Cas9 was performed. The mutant had enhanced salt tolerance without affecting rice growth and development, suggesting that OsCML9 gene is an ideal target gene to generate salt tolerant cultivars by genome editing in the future.

Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

FGF4 protects the liver from immune-mediated injury by activating CaMKKß-PINK1 signal pathway to inhibit hepatocellular apoptosis.

Immune-mediated liver injury (ILI) is a condition where an aberrant immune response due to various triggers causes the destruction of hepatocytes. Fibroblast growth factor 4 (FGF4) was recently identified as a hepatoprotective cytokine; however, its role in ILI remains unclear. In patients with autoimmune hepatitis (type of ILI) and mouse models of concanavalin A (ConA)- or S-100-induced ILI, we observed a biphasic pattern in hepatic FGF4 expression, characterized by an initial increase followed by a return to basal levels. Hepatic FGF4 deficiency activated the mitochondria-associated intrinsic apoptotic pathway, aggravating hepatocellular apoptosis. This led to intrahepatic immune hyper-reactivity, inflammation accentuation, and subsequent liver injury in both ILI models. Conversely, administration of recombinant FGF4 reduced hepatocellular apoptosis and rectified immune imbalance, thereby mitigating liver damage. The beneficial effects of FGF4 were mediated by hepatocellular FGF receptor 4, which activated the Ca2+/calmodulin-dependent protein kinasekinase 2 (CaMKKß) and its downstream phosphatase and tensin homologue-induced putative kinase 1 (PINK1)-dependent B-cell lymphoma 2-like protein 1-isoform L (Bcl-XL) signalling axis in the mitochondria. Hence, FGF4 serves as an early response factor and plays a protective role against ILI, suggesting a therapeutic potential of FGF4 and its analogue for treating clinical immune disorder-related liver injuries.

Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

Rapid sequential clustering of NMDARs, CaMKII, and AMPARs upon activation of NMDARs at developing synapses.

Rapid, synapse-specific neurotransmission requires the precise alignment of presynaptic neurotransmitter release and postsynaptic receptors. How postsynaptic glutamate receptor accumulation is induced during maturation is not well understood. We find that in cultures of dissociated hippocampal neurons at 11 days in vitro (DIV) numerous synaptic contacts already exhibit pronounced accumulations of the pre- and postsynaptic markers synaptotagmin, synaptophysin, synapsin, bassoon, VGluT1, PSD-95, and Shank. The presence of an initial set of AMPARs and NMDARs is indicated by miniature excitatory postsynaptic currents (mEPSCs). However, AMPAR and NMDAR immunostainings reveal rather smooth distributions throughout dendrites and synaptic enrichment is not obvious. We found that brief periods of Ca2+ influx through NMDARs induced a surprisingly rapid accumulation of NMDARs within 1 min, followed by accumulation of CaMKII and then AMPARs within 2-5 min. Postsynaptic clustering of NMDARs and AMPARs was paralleled by an increase in their mEPSC amplitudes. A peptide that blocked the interaction of NMDAR subunits with PSD-95 prevented the NMDAR clustering. NMDAR clustering persisted for 3 days indicating that brief periods of elevated glutamate fosters permanent accumulation of NMDARs at postsynaptic sites in maturing synapses. These data support the model that strong glutamatergic stimulation of immature glutamatergic synapses results in a fast and substantial increase in postsynaptic NMDAR content that required NMDAR binding to PSD-95 or its homologues and is followed by recruitment of CaMKII and subsequently AMPARs.

Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

AIMS: Hyperactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo. METHODS AND RESULTS: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca2+-spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+-leak induction in the CaMKIIδC TG, both regarding Ca2+-spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. CONCLUSIONS: S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

[MiR-132-3p negatively regulates CAMTA1 to promote Schwann cell proliferation and migration and alleviates I-125 seeds-induced exacerbation of facial nerve injury in rats].

OBJECTIVE: To investigate the regulatory effect of miR-132-3p on calmodulin-binding transcription activator 1 (CAMTA1) and Schwann cell activity in rats with facial nerve injury (FNI) treated with I-125 seeds. METHODS: Rat Schwann cells were irradiated with I-125 seeds and transfected with miR-132-3p mimic, miR-132-3p inhibitor or sh-CAMTA1. The expressions of S100B and ß-tubulin Ⅲ in the cells were detected with immunofluorescence assay, and the expressions of miR-132-3p and CAMTA1 protein were determined using RT-qPCR and Western blotting, respectively. EdU staining and Transwell assay were used to evaluate the changes in cell proliferation and migration ability. In a rat model of FNI, I-125 seeds were implanted into the facial tissues near the facial nerve 2 weeks before modeling, and miR-132-3p mimic was injected subcutaneously in the face after modeling. The pathologies of the facial nerve was assessed by HE, LFB and immunofluorescence staining. The targeting relationship between miR-132-3p and CAMTA1 was verified using StarBase v2.0 database and dual-luciferase reporter assay. RESULTS: Rat Schwann cells showed high expressions of S100B and ß-tubulin Ⅲ. I-125 seeds radiation significantly decreased miR-132-3p expression and repressed proliferation and migration of the cells (P < 0.001). Overexpression of miR-132-3p or CAMTA1 knockdown obviously enhanced proliferation and migration of the Schwann cells, while miR-132-3p knockdown produced the opposite effect. MiR-132-3p negatively regulated CAMTA1 expression. In the rat models of FNI, miR-132-3p injection significantly inhibited CAMTA1 expression and attenuated I-125 seeds-induced exacerbation of FNI. CONCLUSION: Overexpression of miR-132-3p suppresses CAMTA1 expression and promotes Schwann cell proliferation and migration to alleviate I-125 seeds-induced exacerbation of FNI in rats.

Functional Analysis of the GhIQD1 Gene in Cotton Resistance to Verticillium Wilt.

Cotton is a critical crop with massive economic implications worldwide. Verticillium wilt is a soil-borne ailment caused by Verticillium dahliae, which harms the growth and development of cotton. Therefore, investigating the genes associated with resistance to verticillium wilt is of particular significance. In this study, we identified the GhIQD1 gene through transcriptome analysis and experimentally characterized the role of the GhIQD1 gene in cotton against V. dahliae. The findings indicated that GhIQD1 acts as a calmodulin-binding protein. The expression of GhIQD1 was the highest in stems, and the expression level increased significantly following infection with V. dahliae. The expression in resistant cotton varieties was higher than in susceptible cotton varieties. Through overexpression of the GhIQD1 gene in tobacco, these transgenic plants exhibited improved resistance to V. dahliae. In contrast, by silencing the GhIQD1 gene in cotton through VIGS, the resistance to V. dahliae was reduced. Following inoculation, the leaves yellowed, and the disease index was higher. Transcriptome analysis of transgenic tobacco 72 h after inoculation indicated that overexpression of GhIQD1 increased the enrichment of the calmodulin pathway and stimulated the production of plant hormones alongside secondary metabolites. Consequently, we investigated the relationship between the GhIQD1 gene and plant disease-resistant hormones SA, JA, and ABA. In summary, this study uncovered the mechanism by which GhIQD1 conferred resistance to V. dahliae in cotton through positive regulation of JA and ABA, providing crucial information for further research on the adaptation of plants to pathogen invasion.

Fluorometric Measurement of Calmodulin-Dependent Peptide-Protein Interactions Using Dansylated Calmodulin.

The assessment of peptide-protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions. Key features • Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding. • Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts. • Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development. • Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.

Fungal alkaloid malbrancheamide reorients the lipid binding domain of GRK5.

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in various types of signaling. GPCR signaling is regulated via receptor phosphorylation by G protein-coupled receptor kinases 5 (GRK5). Calmodulin (CaM), a universal Ca2+ sensor, inhibits receptor phosphorylation by binding to GRK5. However, the inhibitor malbrancheamide (MBC), which binds at CaM C-lobe, allows for receptor phosphorylation. To understand the phosphorylation mechanism by GRK5, we carried out a MD simulation of the CaM/GRK5 complex in the presence and absence of the MBC inhibitor. The lipid binding domain (LBD) of GRK5 adopted different positions in the presence and absence of inhibitor. Furthermore, the inhibitor MBC restricted the movement of the N-lobe tether (NLT) loop, probably blocking the autophosphorylation of GRK5.Communicated by Ramaswamy H. Sarma.

[PI3K/Akt/Erk signaling pathway mediates neuroprotection of CaMK⠡γ and CaMK⠡δ against ischemic reperfusion injury in mice].

OBJECTIVE: To observe neuroprotective effects of Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)γ and CaMkII δ against acute neuronal ischemic reperfusion injury in mice and explore the underlying mechanism. METHODS: Primary cultures of brain neurons isolated from fetal mice (gestational age of 18 days) were transfected with two specific siRNAs (si-CAMK2G and si-CAMK2D) or a control sequence (si-NT). After the transfection, the cells were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) conditions for 1 h followed by routine culture. The expressions of phosphatidylinositol-3-kinase/extracellular signal-regulated kinase (PI3K/Akt/Erk) signaling pathway components in the neurons were detected using immunoblotting. The expressions of the PI3K/Akt/Erk signaling pathway proteins were also detected in the brain tissues of mice receiving middle cerebral artery occlusion (MCAO) or sham operation. RESULTS: The neuronal cells transfected with siCAMK2G showed significantly lower survival rates than those with si-NT transfection at 12, 24, 48, and 72 h after OGD/R (P < 0.01), and si-CAMK2G transfection inhibited OGD/R-induced upregulation of CaMKⅡγ expression. Compared to si-NT, transfection with si-CAMK2G and si-CAMK2D both significantly inhibited the expressions of PI3K/Akt/Erk signaling pathway components (P < 0.01). In the mouse models of MCAO, the expressions of CaMKⅡδ and CaMKⅡγ were significantly increased in the brain, where activation of the PI3K/Akt/Erk signaling pathway was detected. The expression levels of CaMKⅡδ, CaMKⅡγ, Erk, phosphorylated Erk, Akt, and phosphorylated Akt were all significantly higher in MCAO mice than in the sham-operated mice at 24, 48, 72, and 96 h after reperfusion (P < 0.05). CONCLUSION: The neuroprotective effects of CaMKⅡδ and CaMKⅡγ against acute neuronal ischemic reperfusion injury are mediated probably by the PI3K/Akt/Erk pathway.

CAMSAP3-mediated regulation of HMGB1 acetylation and subcellular localization in lung cancer cells: Implications for cell death modulation.

BACKGROUND: Deregulation of cell death is a common characteristic of cancer, and resistance to this process often occurs in lung cancer. Understanding the molecular mechanisms underlying an aberrant cell death is important. Recent studies have emphasized the involvement of calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) in lung cancer aggressiveness, its influence on cell death regulation remains largely unexplored. METHODS: CAMSAP3 was knockout in lung cancer cells using CRISPR-Cas9 system. Cell death and autophagy were evaluated using MTT and autophagic detection assays. Protein interactions were performed by proteomic analysis and immunoprecipitation. Protein expressions and their cytoplasmic localization were analyzed through immunoblotting and immunofluorescence techniques. RESULTS: This study reveals a significant correlation between low CAMSAP3 expression and poor overall survival rates in lung cancer patients. Proteomic analysis identified high mobility group box 1 (HMGB1) as a candidate interacting protein involved in the regulation of cell death. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) resulted in increased HMGB1 acetylation and its translocation to the cytoplasm and secretion, thereby inducing autophagic cell death. However, this process was diminished in CAMSAP3 knockout lung cancer cells. Mechanistically, immunoprecipitation indicated an interaction between CAMSAP3 and HMGB1, particularly with its acetylated form, in which this complex was elevated in the presence of TSA. CONCLUSIONS: CAMSAP3 is prerequisite for TSA-mediated autophagic cell death by interacting with cytoplasmic acetylated HMGB1 and enhancing its release. SIGNIFICANT: This finding provides molecular insights into the role of CAMSAP3 in regulating cell death, highlighting its potential as a therapeutic target for lung cancer treatment.

Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

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.

Genome-Wide Identification of Calmodulin-Binding Protein 60 Gene Family and the Function of GhCBP60B in Cotton Growth and Development and Abiotic Stress Response.

The calmodulin-binding protein 60 (CBP60) family is a gene family unique to plants, and its members play a crucial role in plant defense responses to pathogens and growth and development. Considering that cotton is the primary source of natural cotton textile fiber, the functional study of its CBP60 gene family members is critical. In this research, we successfully identified 162 CBP60 members from the genomes of 21 species. Of these, 72 members were found in four cotton species, divided into four clades. To understand the function of GhCBP60B in cotton in depth, we conducted a detailed analysis of its sequence, structure, cis-acting elements, and expression patterns. Research results show that GhCBP60B is located in the nucleus and plays a crucial role in cotton growth and development and response to salt and drought stress. After using VIGS (virus-induced gene silencing) technology to conduct gene silencing experiments, we found that the plants silenced by GhCBP60B showed dwarf plants and shortened stem nodes, and the expression of related immune genes also changed. In further abiotic stress treatment experiments, we found that GhCBP60B-silenced plants were more sensitive to drought and salt stress, and their POD (peroxidase) activity was also significantly reduced. These results imply the vital role of GhCBP60B in cotton, especially in regulating plant responses to drought and salt stress. This study systematically analyzed CBP60 gene family members through bioinformatics methods and explored in depth the biological function of GhCBP60B in cotton. These research results lay a solid foundation for the future use of the GhCBP60B gene to improve cotton plant type and its drought and salt resistance.

Identification of Riluzole derivatives as novel calmodulin inhibitors with neuroprotective activity by a joint synthesis, biosensor, and computational guided strategy.

The development of new molecules for the treatment of calmodulin related cardiovascular or neurodegenerative diseases is an interesting goal. In this work, we introduce a novel strategy with four main steps: (1) chemical synthesis of target molecules, (2) Förster Resonance Energy Transfer (FRET) biosensor development and in vitro biological assay of new derivatives, (3) Cheminformatics models development and in vivo activity prediction, and (4) Docking studies. This strategy is illustrated with a case study. Firstly, a series of 4-substituted Riluzole derivatives 1-3 were synthetized through a strategy that involves the construction of the 4-bromoriluzole framework and its further functionalization via palladium catalysis or organolithium chemistry. Next, a FRET biosensor for monitoring Ca2+-dependent CaM-ligands interactions has been developed and used for the in vitro assay of Riluzole derivatives. In particular, the best inhibition (80%) was observed for 4-methoxyphenylriluzole 2b. Besides, we trained and validated a new Networks Invariant, Information Fusion, Perturbation Theory, and Machine Learning (NIFPTML) model for predicting probability profiles of in vivo biological activity parameters in different regions of the brain. Next, we used this model to predict the in vivo activity of the compounds experimentally studied in vitro. Last, docking study conducted on Riluzole and its derivatives has provided valuable insights into their binding conformations with the target protein, involving calmodulin and the SK4 channel. This new combined strategy may be useful to reduce assay costs (animals, materials, time, and human resources) in the drug discovery process of calmodulin inhibitors.

Understanding the pathogenic significance of altered calcium-calmodulin signaling in T cells in autoimmune diseases.

Calcium/calmodulin-dependent protein kinase IV (CaMK4) serves as a pivotal mediator in the regulation of gene expression, influencing the activity of transcription factors within a variety of immune cells, including T cells. Altered CaMK4 signaling is implicated in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and psoriasis, which are characterized by dysregulated immune responses and clinical complexity. These conditions share common disturbances in immune cell functionality, cytokine production, and autoantibody generation, all of which are associated with disrupted calcium-calmodulin signaling. This review underscores the consequences of dysregulated CaMK4 signaling across these diseases, with an emphasis on its impact on Th17 differentiation and T cell metabolism-processes central to maintaining immune homeostasis. A comprehensive understanding of roles of CaMK4 in gene regulation across various autoimmune disorders holds promise for the development of targeted therapies, particularly for diseases driven by Th17 cell dysregulation.

Post-translational mechanisms in psychostimulant-induced neurotransmitter efflux.

The availability of monoamine neurotransmitters in the brain is under the control of dopamine, norepinephrine, and serotonin transporters expressed on the plasma membrane of monoaminergic neurons. By regulating transmitter levels these proteins mediate crucial functions including cognition, attention, and reward, and dysregulation of their activity is linked to mood and psychiatric disorders of these systems. Amphetamine-based transporter substrates stimulate non-exocytotic transmitter efflux that induces psychomotor stimulation, addiction, altered mood, hallucinations, and psychosis, thus constituting a major component of drug neurochemical and behavioral outcomes. Efflux is under the control of transporter post-translational modifications that synergize with other regulatory events, and this review will summarize our knowledge of these processes and their role in drug mechanisms.