Search

All biological processes arise through the coordinated actions of biochemical pathways. How such functional diversity is achieved by a finite cast of molecular players remains a central mystery in biology. Spatial compartmentation-the idea that biochemical activities are organized around discrete spatial domains within cells-was first proposed nearly 40 years ago and has become firmly rooted in our understanding of how biochemical pathways are regulated to ensure specificity. However, directly interrogating spatial compartmentation and its mechanistic origins has only really become possible in the last 20 or so years, following technological advances such as the development of genetically encoded fluorescent biosensors. These powerful molecular tools permit a direct, real-time visualization of dynamic biochemical processes in native biological contexts, and they are essential for probing the spatial regulation of biochemical activities. In this Account, we review our labs efforts in developing and using biosensors to map the spatial compartmentation of intracellular signaling pathways and illuminate key mechanisms that establish the boundaries of an intricate biochemical activity architecture. We first discuss the role of regulatory fences, wherein the dynamic activation and deactivation of diffusible messengers produce diverse signaling compartments. For example, we used biosensors for the Ca2+ effector calmodulin and its downstream target calcineurin to reveal a spatial gradient of calmodulin that controls the temporal dynamics of calcineurin signaling. Our studies using cyclic adenosine monophosphate (cAMP) biosensors have similarly elucidated fenced cAMP domains generated by competing production and degradation pathways, ranging in size from cell-spanning gradients to nanoscale hotspots. Second, we describe the role played by intracellular membranes in creating unique signaling platforms with distinctive pathway regulation, as revealed through studies using subcellularly targeted fluorescent biosensors. Using biosensors to visualize subcellular extracellular response kinase (ERK) pathway activity, for example, led us to discover a local signaling circuit that mediates distinct plasma membrane ERK dynamics versus global ERK signaling. Similarly, our work developing biosensors to monitor the subcellular mechanistic target of rapamycin complex 1 (mTORC1) signaling allowed us to not only clarify the presence of mTORC1 activity in the nucleus but also identify a novel mechanism governing the activation of mTORC1 in this location. Finally, we detail how molecular assemblies enable the precise spatial tuning of biochemical activity, through investigations enabled by cutting-edge advances in biosensor design. We recently identified liquid-liquid phase separation as a major factor in cAMP compartmentation aided by a new strategy for targeting biosensors to endogenously expressed proteins via genome editing, for instance, and have also been able to directly visualize nanometer-scale protein kinase signalosomes using an entirely new class of biosensors specifically developed for the dynamic super-resolution imaging of live-cell biochemical activities. Our work provides key insights into the molecular logic of spatially regulated signaling and lays the foundation for a broader exploration of biochemical activity architectures across multiple spatial scales.

[Figure: see text].

Calmodulin (CaM) regulates the activity of a Ca2+ channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca2+-bound CaM bound to RyR2 CaMBD3. The structure reveals Ca2+ bound to the four EF-hands of CaM as well as a fifth Ca2+ bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca2+ and weakens the binding of CaMBD3 to Ca2+-bound CaM. Thus, the binding of the fifth Ca2+ is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca2+ to CaM may contribute to the regulation of RyR2 channel function.

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Small-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

Abstract Background Elongation factor 1 A (EF1A), an essential regulator for protein synthesis, has been reported to participate in abiotic stress responses and environmental adaption in plants. However, the role of EF1A in abiotic stress response was barely studied in Medicago truncatula. Here, we identified elongation factor (EF) genes of M. truncatula and studied the salt stress response function of MtEF1A1 (MTR_6g021805). Results A total of 34 EF genes were identified in the M. truncatula genome. Protein domains and motifs of EFs were highly conserved in plants. MtEF1A1 has the highest expression levels in root nodules and roots, followed by the leaves and stems. Transgenic Arabidopsis thaliana overexpressing MtEF1A1 was more resistant to salt stress treatment, with higher germination rate, longer roots, and more lateral roots than wild type plant. In addition, lower levels of H2O2 and malondialdehyde (MDA) were also detected in transgenic Arabidopsis. Similarly, MtEF1A1 overexpressing M. truncatula was more resistant to salt stress and had lower levels of reactive oxygen species (ROS) in leaves. Furthermore, the expression levels of abiotic stress-responsive genes (MtRD22A and MtCOR15A) and calcium-binding genes (MtCaM and MtCBL4) were upregulated in MtEF1A1 overexpressing lines of M. truncatula. Conclusion These results suggested that MtEF1A1 play a positive role in salt stress regulation. MtEF1A1 may realize its function by binding to calmodulin (CaM) or by participating in Ca2+-dependent signaling pathway. This study revealed that MtEF1A1 is an important regulator for salt stress response in M. truncatula, and provided potential strategy for salt-tolerant plant breeding.

Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.

Activation of Ca2+/calmodulin kinase II (CaMKII) and the N-Methyl D-aspartate receptor (NMDAR), particularly its GluN2B subunit, contribute to the central sensitization of nociceptive pathways and persistent pain. Using mutant mice wherein the activity-driven binding of CaMKII to S1303 in GluN2B is abrogated (GluN2BKI), this study investigated the importance of this interaction for acute and persistent inflammatory nociception. GluN2BKI, wild type and heterozygote mice did not differ in responses to acute noxious heat stimuli as measured with tail flick, paw flick, or hot plate assays, nor did they differ in their responses to mechanical stimulation with von Frey filaments. Surprisingly, the three genotypes exhibited similar spontaneous pain behaviors and hypersensitivity to heat or mechanical stimuli induced by intraplantar injection of capsaicin; however, GluN2BKI mice did not immediately attend to the paw. WT and GluN2BKI mice also did not differ in the nociceptive behaviors elicited by intraplantar injection of formalin, even though MK801 greatly reduced these behaviors in both genotypes concordant with NMDAR dependence. CaMKII binding to GluN2B at S1303 therefore does not appear to be critical for the development of inflammatory nociception. Finally, intrathecal KN93 reduced formalin-induced nociceptive behaviors in GluN2BKI mice. KN93 does not inhibit CaKMII, but rather binds Ca2+/calmodulin. It has multiple other targets including Ca2+-, Na+- and K+-channels, as well as various kinases. Therefore, the use of GluN2BKI mice provided genetic specificity in assessing the role of CaMKII in inflammatory pain signaling cascades. These results challenge current thinking on the involvement of the CaMKII-NMDAR interaction in inflammatory pain.

Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5-4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40-260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.

L-type voltage-gated Ca2+ channels (CaV1.2 and CaV1.3, called CaV) interact with the Ca2+ sensor proteins, calmodulin (CaM) and Ca2+ binding Protein 1 (CaBP1), that oppositely control Ca2+-dependent channel activity. CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca2+ levels (caused by CaV channel opening), Ca2+-bound CaM binding to CaV is essential for promoting rapid Ca2+-dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca2+-induced channel opening (called CDF). In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca2+-free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca2+ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca2+ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca2+ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca2+-bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca2+-bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.

Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.

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.

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.

Explicit description of atomic polarizability is critical for the accurate treatment of inter-molecular interactions by force fields (FFs) in molecular dynamics (MD) simulations aiming to investigate complex electrostatic environments such as metal-binding sites of metalloproteins. Several models exist to describe key monovalent and divalent cations interacting with proteins. Many of these models have been developed from ion–amino-acid interactions and/or aqueous-phase data on cation solvation. The transferability of these models to cation–protein interactions remains uncertain. Herein, we assess the accuracy of existing FFs by their abilities to reproduce hierarchies of thousands of Ca2+–dipeptide interaction energies based on density-functional theory calculations. We find that the Drude polarizable FF, prior to any parameterization, better approximates the QM interaction energies than any of the non-polarizable FFs. Nevertheless, it required improvement in order to address polarization catastrophes where, at short Ca2+–carboxylate distances, the Drude particle of oxygen overlaps with the divalent cation. To ameliorate this, we identified those conformational properties that produced the poorest prediction of interaction energies to reduce the parameter space for optimization. We then optimized the selected cation–peptide parameters using Boltzmann-weighted fitting and evaluated the resulting parameters in MD simulations of the N-lobe of calmodulin. We also parameterized and evaluated the CTPOL FF, which incorporates charge-transfer and polarization effects in additive FFs. This work shows how QM-driven parameter development, followed by testing in condensed-phase simulations, may yield FFs that can accurately capture the structure and dynamics of ion–protein interactions. [ABSTRACT FROM AUTHOR]

IntroductionAn attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists.MethodsIn this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU).ResultsCWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. DiscussionAltogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis

Missense variants in CALM genes encoding the Ca2+-binding protein calmodulin (CaM) cause severe cardiac arrhythmias. The disease mechanisms have been attributed to dysregulation of RyR2, for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) and/or CaV1.2, for Long-QT Syndrome (LQTS). Recently, a novel CALM2 variant, G114R, was identified in a mother and two of her four children, all of whom died suddenly while asleep at a young age. The G114R variant impairs closure of CaV1.2 and RyR2, consistent with a CPVT and/or mild LQTS phenotype. However, the children carrying the CALM2 G114R variant displayed a phenotype commonly observed with variants in NaV1.5, i.e., Brugada Syndrome (BrS) or LQT3, where death while asleep is a common feature. We therefore hypothesized that the G114R variant specifically would interfere with NaV1.5 binding. Here, we demonstrate that CaM binding to the NaV1.5 IQ-domain is severely impaired for two CaM variants G114R and G114W. The impact was most severe at low and intermediate Ca2+ concentrations (up to 4 µM) resulting in more than a 50-fold reduction in NaV1.5 binding affinity, and a smaller 1.5 to 11-fold reduction at high Ca2+ concentrations (25-400 µM). In contrast, the arrhythmogenic CaM-N98S variant only induced a 1.5-fold reduction in NaV1.5 binding and only at 4 µM Ca2+. A non-arrhythmogenic I10T variant in CaM did not impair NaV1.5 IQ binding. These data suggest that the interaction between NaV1.5 and CaM is decreased with certain CaM variants, which may alter the cardiac sodium current, INa. Overall, these results suggest that the phenotypic spectrum of calmodulinopathies may likely expand to include BrS- and/or LQT3-like traits. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD), one of the most prevalent neurodegenerative diseases is associated with pathological autophagy-lysosomal pathway dysfunction. Dexmedetomidine (Dex) has been suggested as an adjuvant to general anesthesia with advantages in reducing the incidence of postoperative cognitive dysfunction in Dex-treated patients with AD and older individuals. Several studies reported that Dex improved memory; however, evidence on the effects of Dex on neuronal autophagy dysfunction in the AD model is lacking. We hypothesized that Dex administration would have neuroprotective effects by improving pathological autophagy dysfunction in mice that received an intracerebroventricular (i.c.v.) injection of amyloid β-protein fragment 25-35 (Aβ25--35) and in an autophagy-deficient cellular model. In the Y-maze test, Dex reversed the decreased activity of Aβ25--35 mice. Additionally, it restored the levels of two memory-related proteins, phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII) and postsynaptic density-95 (PSD-95) in Aβ25-35 mice and organotypic hippocampal slice culture (OHSC) with Aβ25-35. Dex administration also resulted in decreased expression of the autophagy-related microtubule-associated proteins light chain 3-II (LC3-II), p62, lysosome-associated membrane protein2 (LAMP2), and cathepsin D in Aβ25-35 mice and OHSC with Aβ25-35. Increased numbers of co-localized puncta of LC3-LAMP2 or LC3-cathepsin D, along with dissociated LC3-p62 immunoreactivity following Dex treatment, were observed. These findings were consistent with the results of western blots and the transformation of double-membrane autophagosomes into single-membraned autolysosomes in ultrastructures. It was evident that Dex treatment alleviated impaired autolysosome formation in Aβ mice. Our study demonstrated the improvement of memory impairment caused by Dex and its neuroprotective mechanism by investigating the role of the autophagy-lysosomal pathway in a murine Aβ25-35 model. These findings suggest that Dex could be used as a potential neuroprotective adjuvant in general anesthesia to prevent cognitive decline. [ABSTRACT FROM AUTHOR]

The calcium-responsive phosphatase, calcineurin, senses changes in Ca2+ concentrations in a calmodulin-dependent manner. Here we report that under non-stress conditions, inactivation of calcineurin signaling or deleting the calcineurin-dependent transcription factor CRZ1 triggered the formation of chaperone Hsp100p (Hsp104p)-associated protein aggregates in Saccharomyces cerevisiae. Furthermore, calcineurin inactivation aggravated α-Synuclein-related cytotoxicity. Conversely, elevated production of the calcineurin activator, Cnb1p, suppressed protein aggregation and cytotoxicity associated with the familial Parkinson's disease-related mutant α-Synuclein A53T in a partly CRZ1-dependent manner. Activation of calcineurin boosted normal localization of both wild type and mutant α-synuclein to the plasma membrane, an intervention previously shown to mitigate α-synuclein toxicity in Parkinson's disease models. The findings demonstrate that calcineurin signaling, and Ca2+ influx to the vacuole, limit protein quality control in non-stressed cells and may have implications for elucidating to which extent aberrant calcineurin signaling contributes to the progression of Parkinson's disease(s) and other synucleinopathies. 55zLzriqK_eYGg5W2i5yhi Video Abstract [ABSTRACT FROM AUTHOR]

CLICK-III/CaMKIγ is a lipid-anchored neuronal isoform of multifunctional Ca2+/calmodulin-dependent protein kinases, which mediates BDNF-dependent dendritogenesis in cultured cortical neurons. We found that two distinct lipidation states of CaMKIγ, namely, prenylation and palmitoylation, controlled its association with detergent-resistant microdomains in the dendrites and were essential for its dendritogenic activity. However, the impact of each lipid modification on membrane targeting/trafficking and how it specifies functional coupling leading to polarized changes in neuronal morphology are not clear. Here, we show that prenylation induces membrane anchoring of CaMKIγ, permitting access to the Golgi apparatus, and a subsequent palmitoylation facilitates association with cholesterol-enriched lipid microdomains or lipid rafts, in particular at the Golgi. To specifically test the role of palmitoylated CaMKγ in neurite extension, we identified and took advantage of a cell system, PC12, which, unlike neurons, conveniently lacked CaMKIγ and was deficient in the activity-dependent release of a neuritogenic growth factor while possessing the ability to activate polarized rafts signaling for morphogenesis. This system allowed us to rigorously demonstrate that an activity-dependent, lipid rafts-restricted Rae activation leading to neuritogenesis could be functionally rescued by dually lipidated CaMKIγ expression, revealing that not only prenylation but also palmitoylation is essential for CaMKIγ to activate a compartmentalized STEF-Rac1 pathway. These results shed light on the significance of recruiting prenylated and palmitoylated CaMKIγ into the coalescing signalosomes at lipid rafts together with Rac1 and its specific GEF and STEF and forming a compartmentalized Ca2+ signaling pathway that underlies activity-dependent neuritogenesis and morphogenesis during axodendritic polarization critical for brain development and circuitogenesis. [ABSTRACT FROM AUTHOR]

Objective: Lung cancer is prevalent worldwide and a leading contributor to tumor death. This research intends to explore the molecular mechanism of the microRNA‐651‐5p (miR‐651‐5p)/Calmodulin 2 (CALM2) axis in the proliferation, migration, and invasion of lung cancer cells. Methods: Lung cancer tissues and para‐cancerous tissues were collected. The expression levels of miR‐651‐5p and CALM2 in lung cancer tissues and cells were tested, and the connection between miR‐651‐5p expression and clinicopathological characteristics of lung cancer patients was further analyzed. The binding sites between miR‐651‐5p and CALM2 were analyzed and validated. Lung cancer cell proliferation, migration, invasion, and apoptosis were examined. Results: miR‐651‐5p was lowly expressed in lung cancer tissues and cells. miR‐651‐5p expression had no correlation with patients' age and gender but had a correlation with patients' tumor size, TNM stage, and lymph node metastasis. Overexpression of miR‐651‐5p repressed proliferative, migratory, and invasive behaviors of lung cancer cells. miR‐651‐5p targeted and negatively regulated CALM2 expression, and CALM2 reversed the inhibiting effects of miR‐651‐5p on lung cancer cell malignant behaviors, including proliferation, migration, and invasion. Conclusion: This study expounds that miR‐651‐5p affects the proliferation, migration, and invasion of lung cancer cells by regulating CALM2 expression. [ABSTRACT FROM AUTHOR]

The effect of exogenous calmodulin (CaM) on the transport and catalytic activities of Ca2+-ATPase in the symbiosome membrane (SM) from broad bean root nodules was studied at various pH values and concentration of free calcium in the medium (pCa). It was shown that the concentration of H+ ions, which determines the effective interaction of CaM with Ca2+-ATPase, corresponds to pH 7.0, which coincides with the pH value that ensures the optimal functioning of this enzyme. Manifestation of the calmodulin effect was observed only at low Ca2+ concentrations in the medium. The minimum Ca2+ concentration at which CaM initiates stimulation of the ATP-dependent transport of this cation through the SM catalyzed by Ca2+-ATPase is 0.05 μM. Activation of transport activity of Ca2+-ATPase increases with Ca2+-sensor concentration from 20 µg/mL to 80 µg/mL and is at its maximum at a free calcium concentration of 0.118 μM, while hydrolytic activity of the enzyme—at 0.351 µM, when CaM level is 40 µg/mL, which corresponds to half of its saturation dose. Thus, optimal conditions have been identified for the functioning of CaM as a Ca2+-ATPase stimulator, which makes it possible to study the role of this enzyme as an important determinant of Ca2+ signals in the implementation of processes associated with the nitrogen-fixing function of symbiosomes. [ABSTRACT FROM AUTHOR]

In response to Ca2+ signals, the evolutionarily‐conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM‐like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two‐hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin‐binding transcriptional activators (CAMTAs) and myosins, all of which possess tandem isoleucine‐glutamine (IQ) structural domains. Using the split‐luciferase complementation assay in planta and the yeast 2‐hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non‐IQ, CaM‐binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic‐nucleotide gated channel‐20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ‐protein and found that only CaM, CML13 and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13 and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+. Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein‐tagged versions of CaM, CML13 and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co‐expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins. Summary Statement: Plants possess many calmodulin‐like proteins (CMLs), but very few downstream targets of CMLs have been identified. We show that Arabidopsis CML13 and CML14 interact with proteins with multiple IQ‐domain proteins, including CAMTAs, myosins and IQDs, suggesting broad cellular roles for these CMLs. [ABSTRACT FROM AUTHOR]

Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0–10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ−stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV. [ABSTRACT FROM AUTHOR]

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis, CAMTA3 regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLRs (Nucleotide-binding, Leucine-rich repeat Receptors) in modulating plant immunity, raising the possibility that CAMTA3 transcriptional activity is dispensable for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA-binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated six conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA-binding activity. In addition, transient assays showed that these residues are essential for the CAMTA3 function in activating the Rapid Stress Response Element (RSRE)-driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function. [ABSTRACT FROM AUTHOR]

As significant Ca2+ sensors, calmodulin (CaM) and calmodulin-like proteins (CML), have been associated with a variety of environmental conditions in plants. However, whether CaMs/CMLs are related to the stress of phytoplasma infection has not been reported in Paulownia fortunei. In the current study, 5 PfCaMs and 58 PfCMLs were detected through a genome-wide investigation. The number of EF-hand motifs in all PfCaMs/CMLs varied. Bioinformatics analyses, including protein characteristics, conserved domain, gene structure, cis-elements, evolutionary relationship, collinearity, chromosomal location, post-translation modification site, subcellular localization and expression pattern analyses, represented the conservation and divergence of PfCaMs/CMLs. Furthermore, some PfCaMs/CMLs might be involved in plants' reaction to phytoplasma infection and exogenous calcium therapy, indicating these genes may play a role in abiotic as well as biotic stress responses. In addition, subcellular localization analysis showed that PfCML10 was located in the cell membrane and nucleus. In summary, these findings establish a stronger platform for their subsequent functional investigation in trees and further characterize their roles in Paulownia witches' broom (PaWB) occurrence. [ABSTRACT FROM AUTHOR]

OsMADS29 (M29) is a crucial regulator of seed development in rice. The expression of M29 is strictly regulated at transcriptional as well as post‐transcriptional levels. The MADS‐box proteins are known to bind to DNA as dimers. However, in the case of M29, the dimerization also plays a vital role in its localization into the nucleus. The factor(s) that affect oligomerization and nuclear transport of MADS proteins have not yet been characterized. By using BiFC in transgenic BY‐2 cell lines and Yeast‐2‐hybrid assay (Y2H), we show that calmodulin (CaM) interacts with M29 in a Ca2+‐dependent manner. This interaction specifically takes place in the cytoplasm, probably in association with the endoplasmic reticulum. By generating domain‐specific deletions, we show that both sites in M29 are involved in this interaction. Further, by using BiFC‐FRET‐FLIM, we demonstrate that CaM may also help in the dimerization of two M29 monomers. Since most MADS proteins have CaM binding domains, the interaction between these proteins could be a general regulatory mechanism for oligomerization and nuclear transport. [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) is associated with altered cAMP/PKA signaling and an AF-promoting reduction of L-type Ca2+-current (ICa,L), the mechanisms of which are poorly understood. Cyclic-nucleotide phosphodiesterases (PDEs) degrade cAMP and regulate PKA-dependent phosphorylation of key calcium-handling proteins, including the ICa,L-carrying Cav1.2α1C subunit. The aim was to assess whether altered function of PDE type-8 (PDE8) isoforms contributes to the reduction of ICa,L in persistent (chronic) AF (cAF) patients. Methods and results mRNA, protein levels, and localization of PDE8A and PDE8B isoforms were measured by RT-qPCR, western blot, co-immunoprecipitation and immunofluorescence. PDE8 function was assessed by FRET, patch-clamp and sharp-electrode recordings. PDE8A gene and protein levels were higher in paroxysmal AF (pAF) vs. sinus rhythm (SR) patients, whereas PDE8B was upregulated in cAF only. Cytosolic abundance of PDE8A was higher in atrial pAF myocytes, whereas PDE8B tended to be more abundant at the plasmalemma in cAF myocytes. In co-immunoprecipitation, only PDE8B2 showed binding to Cav1.2α1C subunit which was strongly increased in cAF. Accordingly, Cav1.2α1C showed a lower phosphorylation at Ser1928 in association with decreased ICa,L in cAF. Selective PDE8 inhibition increased Ser1928 phosphorylation of Cav1.2α1C, enhanced cAMP at the subsarcolemma and rescued the lower ICa,L in cAF, which was accompanied by a prolongation of action potential duration at 50% of repolarization. Conclusion Both PDE8A and PDE8B are expressed in human heart. Upregulation of PDE8B isoforms in cAF reduces ICa,L via direct interaction of PDE8B2 with the Cav1.2α1C subunit. Thus, upregulated PDE8B2 might serve as a novel molecular mechanism of the proarrhythmic reduction of ICa,L in cAF. [ABSTRACT FROM AUTHOR]

Neurobiologists widely use green genetically encoded calcium indicators (GECIs) for visualization of neuronal activity. Among them, ratiometric GECIs allow imaging of both active and non-active neuronal populations. However, they are not popular, since their properties are inferior to intensiometric GCaMP series of GECIs. The most characterized and developed ratiometric green GECI is FGCaMP7. However, the dynamic range and sensitivity of its large Stock's shift green (LSS-Green) form is significantly lower than its Green form and its molecular design is not optimal. To address these drawbacks, we engineered a ratiometric green calcium indicator, called FNCaMP, which is based on bright mNeonGreen protein and calmodulin from A. niger and has optimal NTnC-like design. We compared the properties of the FNCaMP and FGCaMP7 indicators in vitro, in mammalian cells, and in neuronal cultures. Finally, we obtained and analyzed X-ray structure of the FNCaMP indicator. • A new ratiometric green calcium indicator NCaMP allows visualization of neurons both at rest and during their activity. • Crystal structure of the ratiometric NCaMP calcium indicator reveals key features of its function. • NTnC-like design of FNCaMP ensures that its properties are preserved in fusion constructs with other proteins. • Calmodulin from the A. niger fungus ensures the inertness of FNCaMP to components of the intracellular environment. [ABSTRACT FROM AUTHOR]

Introduction: Previous studies have shown that converting curcumin into nanoparticles can enhance its therapeutic effect in models of convulsion. Changes in calmodulin-dependent Ca2+/protein kinase (CaMKII) are associated with some experimental models of epilepsy. This study aimed to evaluate the hippocampal CaMKII phosphorylation following the therapeutic effect of nanocurcumin in a model of pentylenetetrazole (PTZ)-induced convulsion. Methods: Nanocurcumin (with an average particle diameter of 150 nm) was produced using bovine serum albumin and was administered orally (gavage) to male NMRI mice weighing 25-30 g at doses of 50 and 100 mg/kg. One hour later, PTZ at a dose of 85 mg/kg was administered intraperitoneally to the animal. Following intraperitoneal PTZ injection, the latency of myoclonus, clonus, general tonic convulsion, and mortality rate were recorded in male mice. After behavioral testing, the anesthetized animals were euthanized, and the levels of phosphorylated and total forms of hippocampal CaMKII were determined by Western blotting technique. Results: The results showed that oral administration of nanocurcumin at doses of 50 and 100 mg/kg significantly improved convulsion induced by PTZ compared with natural curcumin. In all groups, convulsion increased hippocampal CaMKII phosphorylation, and nanocurcumin did not modulate it. Conclusion: The findings of this study showed that the therapeutic effect of nanocurcumin in a model of pentylenetetrazoleinduced convulsion was not associated with a change in hippocampal CaMKII activity pattern. [ABSTRACT FROM AUTHOR]

Simple Summary: The aim of the study was to detect the nucleotide sequence variability in the calcium/calmodulin-dependent serine kinase (CASK) gene in pigeons. One of the detected polymorphic sites (g.8893G > A) located at position −3 relative to the start codon was selected for genotyping in 517 pigeons and was used to establish possible associations between genotypes and flight performance of pigeons. Frequencies of the AA genotype were higher in homing pigeon groups than in the group of non-homing pigeons. The results can be treated as a contribution to further research that could confirm the functionality of the analysed SNP in shaping the sports phenotype of domestic pigeons. Calcium/calmodulin-dependent serine protein kinase (CASK) is an multidomain protein involved in tissue development and cell signalling. In skeletal muscle, it is involved in the development of neuromuscular junctions. The participation of a pigeon in racing is a great physical effort that causes many changes in the skeletal muscles. Thus, the purpose of the study was to detect the nucleotide sequence variability in the calcium/calmodulin-dependent serine kinase (CASK) gene in domestic pigeons (Columba livia domestica) and assess the potential impact of DNA polymorphisms on the flight performance of pigeons. The research included a total of 517 individuals. DNA was extracted from the blood. A DNA fragment from nucleotides 8689 to 9049 of the CASK (NW_004973256.1 sequence) of six unrelated pigeons were sequenced. One of the detected polymorphic sites (g.8893G > A), located a very close to the start codon, was selected for genotyping in all individuals. The association studies included a total of 311 young homing pigeons that participated in racing competitions. The homing pigeons showed higher frequencies of the AA genotype than non-homing ones (p < 0.05). In rock pigeons only the GG genotype was found. Further research could confirm the functionality of the CASK g.8893G > A SNP in shaping the racing phenotype of pigeons, and the AA genotype could be useful as a selection criterion in pigeon breeding. [ABSTRACT FROM AUTHOR]

Optimizing physical training regimens to increase muscle aerobic capacity requires an understanding of the internal processes that occur during exercise that initiate subsequent adaptation. During exercise, muscle cells undergo a series of metabolic events that trigger downstream signaling pathways and induce the expression of many genes in working muscle fibers. There are a number of studies that show the dependence of changes in the activity of AMP-activated protein kinase (AMPK), one of the mediators of cellular signaling pathways, on the duration and intensity of single exercises. The activity of various AMPK isoforms can change in different directions, increasing for some isoforms and decreasing for others, depending on the intensity and duration of the load. This review summarizes research data on changes in the activity of AMPK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and other components of the signaling pathways in skeletal muscles during exercise. Based on these data, we hypothesize that the observed changes in AMPK activity may be largely related to metabolic and signaling transients rather than exercise intensity per se. Probably, the main events associated with these transients occur at the beginning of the exercise in a time window of about 1–10 min. We hypothesize that these transients may be partly due to putative trigger-like kinase/protein phosphatase interactions regulated by feedback loops. In addition, numerous dynamically changing factors, such as [Ca2+], metabolite concentration, and reactive oxygen and nitrogen species (RONS), can shift the switching thresholds and change the states of these triggers, thereby affecting the activity of kinases (in particular, AMPK and CaMKII) and phosphatases. The review considers the putative molecular mechanisms underlying trigger-like interactions. The proposed hypothesis allows for a reinterpretation of the experimental data available in the literature as well as the generation of ideas to optimize future training regimens. [ABSTRACT FROM AUTHOR]

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between −1519 bp and −589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation. [ABSTRACT FROM AUTHOR]

Porous silicon (PSi) gained a lot of significance lately as a biosensor due to its significantly high absorption capacity, easily modulable porosity, and its biocompatibility. In this work, the etching time dependence of the optical response of PSi-based calmodulin (CaM) surface-functionalized calcium detector is studied. PSi was fabricated on P-type silicon wafers by electrochemical etching process. Increase in electrochemical etching time significantly increases the porosity of the PSi substrate. Thus, the optical response of the PSi-based calcium detector is observed to be remarkably affected by the change in etching time. Studies were performed on both nano (structure size ranging from 6 to 19 nm) and macro (pore size ranging from 6 to 1.5 µm) PSi. Optical parameters such as reflectance, scattering, and absorptance of the PSi-based optical detector were analyzed and notable changes were recorded for changing etching time in both nano- and macro-PSi samples. This study helps to drastically improve the sensitivity of PSi-based optical calcium detector, and the concept may be incorporated in the designing of calcium or other ionic and chemical sensors with improved selectivity and sensitivity. Thus, the study is useful in the fabrication of PSi-based calcium sensors with optimized etching time so that highly sensitive and precise calcium detection can be possible using it. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? SUMMARY ANSWER Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. WHAT IS KNOWN ALREADY The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. STUDY DESIGN, SIZE, DURATION Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn−/−) mouse model was generated by CRISPR–Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography–mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. MAIN RESULTS AND THE ROLE OF CHANCE Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn−/− male mice. VSL, VCL, and VAP in the sperm of Iqcn−/− male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn−/− male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. LIMITATIONS, REASONS FOR CAUTION More cases are needed to demonstrate the relation between IQCN variants and phenotypes. WIDER IMPLICATIONS OF THE FINDINGS Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Molecular genetic analyses in the model species Arabidopsis thaliana have demonstrated the major roles of different CALMODULIN‐BINDING PROTEIN 60 (CBP60) proteins in growth, stress signaling, and immune responses. Prominently, CBP60g and SARD1 are paralogous CBP60 transcription factors that regulate numerous components of the immune system, such as cell surface and intracellular immune receptors, MAP kinases, WRKY transcription factors, and biosynthetic enzymes for immunity‐activating metabolites salicylic acid (SA) and N‐hydroxypipecolic acid (NHP). However, their function, regulation, and diversification in most species remain unclear. Here, we have created CBP60‐DB (https://cbp60db.wlu.ca/), a structural and bioinformatic database that comprehensively characterized 1052 CBP60 gene homologs (encoding 2376 unique transcripts and 1996 unique proteins) across 62 phylogenetically diverse genomes in the plant kingdom. We have employed deep learning‐predicted structural analyses using AlphaFold2 and then generated dedicated web pages for all plant CBP60 proteins. Importantly, we have generated a novel clustering visualization algorithm to interrogate kingdom‐wide structural similarities for more efficient inference of conserved functions across various plant taxa. Because well‐characterized CBP60 proteins in Arabidopsis are known to be transcription factors with putative calmodulin‐binding domains, we have integrated external bioinformatic resources to analyze protein domains and motifs. Collectively, we present a plant kingdom‐wide identification of this important protein family in a user‐friendly AlphaFold‐anchored database, representing a novel and significant resource for the broader plant biology community. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the possible mechanisms by which orphan G protein‐coupled receptor GPR41 activation enhances glucose uptake into C2C12 myotubes using a GPR41‐selective agonist, AR420626, and to examine the ability of this agent to improve insulin sensitivity and glucose homeostasis in vivo. Methods: Basal and insulin‐stimulated glucose uptake and glucose transporter 4 translocations were measured in C2C12 myotubes. Ca2+ influx into cells was measured and GPR41‐mediated signaling by AR420626 was examined. An oral glucose tolerance test was performed, and plasma insulin levels were measured in streptozotocin‐treated or high‐fat diet‐fed diabetic mice. The glycogen content was measured in skeletal muscle tissue. Results: AR420626 increased basal and insulin‐stimulated glucose uptake, which was reduced by pertussis toxin, an inhibitor of Gαi‐mediated signaling, and treatment with small interfering RNA for GPR41 (siGPR41). AR420626 increased intracellular Ca2+ influx and phosphorylated Ca2+/calmodulin‐dependent protein kinase type II, cyclic AMP‐responsive element‐binding protein, and mitogen‐activated protein kinase (p38) in C2C12 myotubes, which were inhibited by treating with pertussis toxin, amlodipine (Ca2+ channel blocker), and siGPR41. AR420626 increased plasma insulin levels and skeletal muscle glycogen content and improved glucose tolerance in streptozotocin‐ and high‐fat diet‐induced diabetic mouse models. Conclusions: GPR41 activation with AR420626 increased glucose uptake mediated by Ca2+ signaling via GPR41, improving diabetes mellitus. [ABSTRACT FROM AUTHOR]

Lithium salts have been the mainstay of treatment for bipolar disorder for more than 50 years, since approval by the FDA in 1970 for the treatment of this pathology. A variety of the molecular mechanisms of action of lithium have been well studied, primarily inhibition of the enzymes glycogen synthase 3β and inositol monophosphatase, with subsequent activation of cascades of cellular reactions, including induction of brain-derived neurotrophic factor and antiapoptotic proteins and suppression of calcium-dependent activation of apoptosis. Research over the last decade has focused on the effects of lithium on the regulation of autophagy and the accumulation of pathological proteins such as amyloid β and tau protein in neurons. Lithium is also thought to induce telomere elongation and to increase telomerase activity. Clinical studies of lithium have addressed the potential for its use for the prevention and treatment of neurodegenerative diseases, primarily Alzheimer's disease, with increasing emphasis on the use of lithium microdoses. A separate scientific problem is the search for safe and effective lithium salts using methods including chemoreactome analysis. [ABSTRACT FROM AUTHOR]

Background: Insulin resistance (IR) is an important determinant of glucose metabolic disturbance and placental dysplasia in gestational diabetes mellitus (GDM). Calcium/calmodulin dependent protein kinase IV (CAMK4) improves insulin IR induced by a high-fat diet (HFD). The current study sought to elucidate the role and potential mechanism of CAMK4 in GDM. Methods: A GDM model was established in female C57BL/6J mice via HFD feeding for one week before mating and throughout gestation. The IR was elicited by 10–6 M insulin treatment for 48 h in HTR-8/SVneo cells and mouse primary trophoblast cells. The function of CAMK4 was investigated by transfection of overexpression plasmid in HTR-8/SVneo cells and infection of lentivirus loaded with CAMK4 encoding sequence in primary trophoblast cells. Real-time PCR, western blot, cell counting kit-8, transwell, wound healing, dual-luciferase reporter assay, and liquid chromatography/mass spectrometry-based untargeted metabolomics were performed to confirm the effects of CAMK4 on trophoblast cells. Results: Decreased CAMK4 expression was found in the placenta of GDM mice. CAMK4 overexpression ameliorated IR-induced viability impairment, migratory and invasive capacity inhibition, autophagy blocking, insulin signaling inactivation and glucose uptake disorder in trophoblast cells. CAMK4 also transcriptionally activated orphan nuclear receptor NUR77, and the effects of CAMK4 were abrogated by silencing of NUR77. Metabolomics analysis revealed that CAMK4 overexpression caused alterations of amino acid, lipid and carbohydrate metabolism, which were important in GDM. Conclusion: Our results indicated that CAMK4/NUR77 axis may provide novel potential targets in GDM treatment. [ABSTRACT FROM AUTHOR]

Background: Electroacupuncture (EA) is a complementary and alternative therapy which has shown protective effects on vascular cognitive impairment (VCI). However, the underlying mechanisms are not entirely understood. Methods: Rat models of VCI were established with cerebral ischemia using occlusion of the middle cerebral artery or bilateral common carotid artery. The brain structure and function imaging were measured through animal MRI. miRNA expression was detected by chip and qPCR. Synaptic functional plasticity was detected using electrophysiological techniques. Results: This study demonstrated the enhancement of Regional Homogeneity (ReHo) activity of blood oxygen level-dependent (BOLD) signal in the entorhinal cortical (EC) and hippocampus (HIP) in response to EA treatment. miR-219a was selected and confirmed to be elevated in HIP and EC in VCI but decreased after EA. N-methyl-D-aspartic acid receptor1 (NMDAR1) was identified as the target gene of miR-219a. miR-219a regulated NMDAR-mediated autaptic currents, spontaneous excitatory postsynaptic currents (sEPSC), and long-term potentiation (LTP) of the EC-HIP CA1 circuit influencing synaptic plasticity. EA was able to inhibit miR-219a, enhancing synaptic plasticity of the EC-HIP CA1 circuit and increasing expression of NMDAR1 while promoting the phosphorylation of downstream calcium/calmodulin-dependent protein kinase II (CaMKII), improving overall learning and memory in VCI rat models. Conclusion: Inhibition of miR-219a ameliorates VCI by regulating NMDAR-mediated synaptic plasticity in animal models of cerebral ischemia. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration–approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications. Editor's summary: Excessive Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity has been associated with a number of cardiovascular diseases, including arrhythmias, but no CaMKII inhibitors have been approved for clinical use because of low efficacy and concerns regarding potential side effects on cognition. Here, Reyes Gaido and colleagues generated a fluorescent reporter of CaMKII activity to screen FDA-approved compounds. They identified JAK1/2 inhibitor ruxolitinib as a potent CaMKII inhibitor and demonstrated that pretreatment with this compound could effectively reduce arrythmias in vitro and in multiple mouse models without negative impacts on learning and memory. These results suggest that ruxolitinib could potentially be further developed as a treatment for certain arrhythmogenic conditions. —Allison Williams [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is frequently associated with β-adrenergic stimulation, especially in patients with structural heart diseases. The objective of this study was to determine the synergism of late sodium current (late INa) and Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated arrhythmogenic activities in β-adrenergic overactivation-associated AF. Monophasic action potential, conduction properties, protein phosphorylation, ion currents and cellular trigger activities were measured from rabbit-isolated hearts, atrial tissue and atrial myocytes, respectively. Isoproterenol (ISO, 1–15 nM) increased atrial conduction inhomogeneity index, phospho-Nav1.5 and phospho-CaMKII protein levels and late INa by 108%, 65%, 135% and 87%, respectively, and induced triggered activities and episodes of AF in all hearts studied (p < 0.05). Sea anemone toxin II (ATX-II, 2 nM) was insufficient to induce any atrial arrhythmias, whereas the propensities of AF were greater in hearts treated with a combination of ATX-II and ISO. Ranolazine, eleclazine and KN-93 abolished ISO-induced AF, attenuated the phosphorylation of Nav1.5 and CaMKII, and reversed the increase of late INa (p < 0.05) in a synergistic mode. Overall, late INa in association with the activation of CaMKII potentiates β-adrenergic stimulation-induced AF and the inhibition of both late INa and CaMKII exerted synergistic anti-arrhythmic effects to suppress atrial arrhythmic activities associated with catecholaminergic activation. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'. [ABSTRACT FROM AUTHOR]

Simple Summary: Trafficking chaperones facilitate the spatio-temporal distribution pattern of proteins inside cells. In the case of the membrane-anchored protein Ras, trafficking chaperones typically bind to the C-terminal farnesyl-moiety. Thus shielded from the aqueous environment, Ras can diffuse more efficiently through the cytoplasm. The calcium-binding protein calmodulin (CaM) was proposed as a K-Ras trafficking chaperone. However, CaM has many different functions inside the cell. Centrin proteins are highly related to calmodulin, and we find that they also bind to K-Ras. Unexpectedly, this interaction depends on the activation state and the effector binding site of K-Ras, not on the farnesyl-anchor. Overall, CaM and centrin1 appear to enable only a fraction of K-Ras membrane anchorage. Given that CaM inhibitors also affect the K-Ras/centrin1 interaction and the very similar distribution of centrin1 and CaM throughout the cell cycle, the dependence of K-Ras on either protein may be difficult to determine. Recent data suggest that K-Ras4B (hereafter K-Ras) can drive cancer cell stemness via calmodulin (CaM)-dependent, non-canonical Wnt-signalling. Here we examined whether another Ca2+-binding protein, the CaM-related centrin1, binds to K-Ras and could mediate some K-Ras functions that were previously ascribed to CaM. While CaM and centrin1 appear to distinguish between peptides that were derived from their classical targets, they both bind to K-Ras in cells. Cellular BRET- and immunoprecipitation data suggest that CaM engages more with K-Ras than centrin1 and that the interaction with the C-terminal membrane anchor of K-Ras is sufficient for this. Surprisingly, binding of neither K-Ras nor its membrane anchor alone to CaM or centrin1 is sensitive to inhibition of prenylation. In support of an involvement of the G-domain of K-Ras in cellular complexes with these Ca2+-binding proteins, we find that oncogenic K-RasG12V displays increased engagement with both CaM and centrin1. This is abrogated by addition of the D38A effector-site mutation, suggesting that K-RasG12V is held together with CaM or centrin1 in complexes with effectors. When treated with CaM inhibitors, the BRET-interaction of K-RasG12V with centrin1 was also disrupted in the low micromolar range, comparable to that with CaM. While CaM predominates in regulating functional membrane anchorage of K-Ras, it has a very similar co-distribution with centrin1 on mitotic organelles. Given these results, a significant overlap of the CaM- and centrin1-dependent functions of K-Ras is suggested. [ABSTRACT FROM AUTHOR]

The phenomena of ischemia and reperfusion are associated with the pathological background of cardiovascular diseases. Ischemia is initiated by ischemia reperfusion injury (IRI), which involves disruption of intracellular signaling pathways and causes cell death. The aim of this study was to assess the reactivity of vascular smooth muscle cells in the conditions of induced ischemia and reperfusion, and to determine the mechanisms leading to contractility disorders. This study was conducted using classical pharmacometric methods on an isolated model of the rat caudal artery. The experiment consisted of the analysis of the final and initial perfusate pressure measurements after induction of arterial contraction with phenylephrine in the presence of forskolin and A7 hydrochloride, two ligands modifying the contractility of vascular smooth muscle cells (VSMC). The pharmacometric analysis showed that in simulated reperfusion, cyclic nucleotides have a vasoconstrictive effect, and calmodulin has a vasodilating effect. The responsiveness of vascular smooth muscle cells to the vasopressor effects of α1-adrenomimetics during reperfusion may change uncontrollably, and the effects of secondary messengers may be counter physiological. Further studies are needed to evaluate the function of other second messengers on VSMCs in the process of ischemia and reperfusion. [ABSTRACT FROM AUTHOR]

SUMMARY: Protein lysine acetylation is an important post‐translational modification mechanism involved in cellular regulation in eukaryotes. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotes and is crucial for plant immunity, but it is so far unclear whether acetylation is involved in CaM‐mediated plant immunity. Here, we found that GhCaM7 is acetylated upon Verticillium dahliae (V. dahliae) infection and a positive regulator of V. dahliae resistance. Overexpressing GhCaM7 in cotton and Arabidopsis enhances V. dahliae resistance and knocking‐down GhCaM7 makes cotton more susceptible to V. dahliae. Transgenic Arabidopsis plants overexpressing GhCaM7 with mutation at the acetylation site are more susceptible to V. dahliae than transgenics overexpressing the wild‐type GhCaM7, implying the importance of the acetylated GhCaM7 in response to V. dahliae infection. Yeast two‐hybrid, bimolecular fluorescent complementation, luciferase complementation imaging, and coimmunoprecipitation assays demonstrated interaction between GhCaM7 and an osmotin protein GhOSM34 that was shown to have a positive role in V. dahliae resistance. GhCaM7 and GhOSM34 are co‐localized in the cell membrane. Upon V. dahliae infection, the Ca2+ content reduces almost instantly in plants with downregulated GhCaM7 or GhOSM34. Down regulating GhOSM34 enhances accumulation of Na+ and increases cell osmotic pressure. Comparative transcriptomic analyses between cotton plants with an increased or reduced expression level of GhCaM7 and wild‐type plants indicate the involvement of jasmonic acid signaling pathways and reactive oxygen species in GhCaM7‐enabled disease resistance. Together, these results demonstrate the involvement of CaM protein in the interaction between cotton and V. dahliae, and more importantly, the involvement of the acetylated CaM in the interaction. Significance Statement: GhCaM7 positively regulator cotton defense responses to Verticillium dahliae by acetylation and interacting with osmotin protein GhOSM34, leading to activation of Ca2+, jasmonic acid, and reactive oxygen species signaling pathways. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro , which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity. • Expression and purification of biotinylation enzyme fused with calmodulin. • AirID-CaM is capable of biotinylating multiple CaM kinases in a Ca2+-dependent manner. • Biotinylation of CaMKKα/1 by AirID-CaM likely occurs in interaction-dependent manner. • AirID-CaM can be useful for the rapid detection of CaM/target interaction. [ABSTRACT FROM AUTHOR]

Background: Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). Methods: Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. Results: CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. Conclusion: These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA; 22:6n-3), an n-3 polyunsaturated fatty acid, has various important roles in brain functions. Nitric oxide (NO) produced by neuronal NO synthase (nNOS) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) is also involved in brain functions. We investigated the influence of DHA on nNOS and CaMKII protein expression in differentiated NG108-15 cells. NG108-15 cells were seeded in 12-well plates, and after 24 h, the medium was replaced with Dulbecco's modified Eagle's medium containing 1% fetal bovine serum, 0.2 mM dibutyryl cyclic adenosine monophosphate and 100 nM dexamethasone as differentiation-inducing medium. When cells were cultured in differentiation-inducing medium, neurite-like outgrowths were observed on days 5 and 6. However, no significant difference in morphology was observed in cells with or without DHA treatment. With or without DHA addition, nNOS protein expression was increased on days 5 and 6 compared with day 0. This increase tended to be enhanced by DHA. CaMKII protein expression did not change after differentiation without DHA, but was significantly increased on day 6 compared with day 0 with DHA addition. These data indicate that DHA is involved in brain functions by regulating CaMKII and nNOS protein expression. [ABSTRACT FROM AUTHOR]

Various protein kinases including protein kinase A (PKA), Ca2+-calmodulin kinase (CaMK), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK: ERK1/2, p38-MAPK and JNK) are integral part of different signal transduction pathways, which are known to regulate cardiac structure, function and metabolism. In addition, these signal transducing proteins are involved in the regulation of cation transport, cellular growth, gene expression, apoptosis and fibrosis by modifying the function of different target sites of subcellular organelles in the myocardium. However, the information regarding these signal transducing molecules is scattered and mechanisms of their involvement in diverse regulatory processes are poorly understood. While PKA, CaMK, PI3K and PKC are activated by different hormones and mechanical stimuli, MAPKs are activated by growth factors and some cellular stresses such as oxidative stress, inflammation and Ca2+-overload. Each type of these protein kinases is expressed in the form of two or more isozymes showing different biochemical characteristics and distinct biological functions. It has been demonstrated that all specific isoforms of these kinases produce both beneficial and detrimental effects on the heart, which are dependent upon the intensity and duration of stimulus for their activation. While PKA, PKC and CaMK are mainly involved in augmenting cardiac function as well as inducing cardiac hypertrophy and arrhythmias, PI3K is mainly involved in maintaining β-adrenoceptor function and inducing inflammation as well as arrhythmias. On the other hand, ERK1/2 mainly participate in the genesis of cardiac hypertrophy and cytoprotection whereas p38-MAPK and JNK are primarily involved in cardiac dysfunction, apoptosis and fibrosis. Since the activities of most protein kinases are increased under prolonged pathological conditions, a wide variety of their inhibitors have been shown to produce beneficial effects. However, extensive research needs to be carried out to understand the pathophysiology of different isoforms of each protein kinase as well as for the development of their isoform-specific inhibitors. [ABSTRACT FROM AUTHOR]

The calmodulin-activated a-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, highthroughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists. Principal among these is the ATP-competitive pyrido-pyrimidinedione inhibitor, A-484954, which shows high specificity toward eEF-2K relative to a panel of "typical" protein kinases. A-484954 has been shown to have some degree of efficacy in animal models of several disease states. It has also been widely deployed as a reagent in eEF-2K-specific biochemical and cellbiological studies. However, given the absence of structural information, the precise mechanism of the A-484954-mediated inhibition of eEF-2K has remained obscure. Leveraging our identification of the calmodulin-activatable catalytic core of eEF-2K, and our recent determination of its long-elusive structure, here we present the structural basis for its specific inhibition by A-484954. This structure, which represents the first for an inhibitor-bound catalytic domain of a member of the a-kinase family, enables rationalization of the existing structure-activity relationship data for A-484954 variants and lays the groundwork for further optimization of this scaffold to attain enhanced specificity/potency against eEF-2K. [ABSTRACT FROM AUTHOR]

Neuropathic pain (NP) is well-known to be complicated and challenging to manage. Organisms have been shown to initiate various intrinsic compensatory strategies to alleviate NP, which could be utilized to aid the development of therapeutic treatments. However, these mechanisms that alleviate NP have not been fully elucidated. Here we show that spinal glycine receptor a1 (GlyRa1) is a key element of an intrinsic compensatory mechanism to alleviate NP. This spinal GlyRa1-mediated compensatory mechanism is manifested in many ways, including enhanced inhibitory glycinergic transmission and increased GlyRa1 protein expression levels. We show such GlyRa1 upregulation is achieved via an intracellular calmodulin kinase II/IV (CaMKII/IV)-cAMP response element binding protein signaling pathway. We demonstrate that dehydroxylcannabidiol, a synthetic nonpsychoactive cannabinoid that specifically targets GlyRs, significantly potentiates spinal GlyR function, inhibits spared nerve injury-induced spinal neuronal hyperactivity, and ultimately abrogates the development of hyperalgesia. These results suggest that the compensatory upregulated spinal GlyRa1 is efficacious in alleviating NP. Thus, our study reveals a potential strategy for the clinical treatment of NP by utilizing this central compensatory mechanism. [ABSTRACT FROM AUTHOR]