Calmodulin-dependent Kinase Research Articles

Importance of Zinc Homeostasis for Normal Cardiac Rhythm

<p>Current arrhythmia therapies such as ion channel blockers, catheter ablation, or implantable cardioverter defibrillators have limitations and side effects, and given the proarrhythmic risk associated with conventional, ion channel-targeted anti-arrhythmic drug therapies, a new approach to arrhythmias may be warranted. Measuring and adjusting the level of particular ions that impact heart rhythm can be a simple and low-complication strategy for preventing or treating specific arrhythmias. In addition, new medicines targeting these ions may effectively treat arrhythmias. Numerous studies have shown that intracellular and extracellular zinc concentrations impact the heart's electrical activity. Zinc has been observed to affect cardiac rhythm through a range of mechanisms. These mechanisms encompass the modulation of sodium, calcium, and potassium ion channels, as well as the influence on beta-adrenergic receptors and the enzyme adenylate cyclase. </p><p> Moreover, zinc can either counteract or induce oxidative stress, hinder calmodulin or the enzyme Ca (2+)/calmodulin-dependent protein kinase II (CaMKII), regulate cellular ATP levels, affect the processes of aging and autophagy, influence calcium ryanodine receptors, and control cellular inflammation. Additionally, zinc has been implicated in the modulation of circadian rhythm. Additionally, zinc has been implicated in the modulation of circadian rhythm. In all the above cases, the effect of zinc largely depends on the normal or increased cellular level of zinc, which shows the importance of maintaining the serum and intracellular levels of zinc within the normal range.</p>

Research advances on CaMKs-mediated neurodevelopmental injury.

Calcium/calmodulin-dependent protein kinases (CaMKs) are important proteins in the calcium signaling cascade response pathway, which can broadly regulate biological functions in vivo. Multifunctional CaMKs play key roles in neural development, including neuronal circuit building, synaptic plasticity establishment, and neurotrophic factor secretion. Currently, four familial proteins, calcium/calmodulin-dependent protein kinase I (CaMKI), calcium/calmodulin-dependent protein kinase II (CaMKII), eukaryotic elongation factor 2 kinase (eEF2K, popularly known as CaMKIII) and calcium/calmodulin-dependent protein kinase IV (CaMKIV), are thought to have been the most extensively studied during neurodevelopment. Although their spatial structures are extremely similar, as well as the initial starting point of activation, both require the activation of calcium and calmodulin (CaM) complexes to be involved in the process, and the phosphorylation sites and modes of each member are different. Furthermore, due to the high structural similarity of CaMKs, their members may play synergistic roles in the regulation of neural development, but different CaMKs also have their own means of regulating neural development. In this review, we first describe the visualized protein structural forms of CaMKI, CaMKII, eEF2K and CaMKIV, and then describe the functions of each kinase in neurodevelopment. After that, we focus on four main mechanisms of neurodevelopmental damage caused by CaMKs: CaMKI/ERK/CREB pathway inhibition leading to dendritic spine structural damage; Ca2+/CaM/CaMKII through induction of mitochondrial kinetic disorders leading to neurodevelopmental damage; CaMKIII/eEF2 hyperphosphorylation affects the establishment of synaptic plasticity; and CaMKIV/JNK/NF-κB through induction of an inflammatory response leading to neurodevelopmental damage. In conclusion, we briefly discuss the pathophysiological significance of aberrant CaMK family expression in neurodevelopmental disorders, as well as the protective effects of conventional CaMKII and CaMKIII antagonists against neurodevelopmental injury.

PSD-95 Protein: A Promising Therapeutic Target in Chronic Pain.

Chronic pain, as a social public health problem, has a serious impact on the quality of patients' life. Currently, the main drugs used to treat chronic pain are opioids, antipyretic, and nonsteroidal anti-inflammatory drugs (NSAIDs). But the obvious side effects limit their use, so it is urgent to find new therapeutic targets. Postsynaptic density (PSD)-95 protein plays an important role in the occurrence and development of chronic pain. The over-expression of the PSD-95 protein and its interaction with other proteins are closelyrelated to the chronic pain. Besides, the PSD-95-related drugs that inhibit the expression of PSD-95 as well as the interaction with other protein have been proved to treat chronic pain significantly. In conclusion, although more deep studies are needed in the future, these studies indicate that PSD-95 and the related proteins, such as NMDA receptor (NMDAR) subunit 2B (GluN2B), AMPA receptor (AMPAR), calmodulin-dependent protein kinase II (CaMKII), 5-hydroxytryptamine 2A receptor (5-HT2AR), and neuronal nitric oxide synthase (nNOS), are the promising therapeutic targets for chronicpain.

TRPC3 suppression ameliorates synaptic dysfunctions and memory deficits in Alzheimer's disease.

Transient receptor potential canonical (TRPC) channels are widely expressed in the brain; however, their precise roles in neurodegeneration, such as Alzheimer's disease (AD) remain elusive. Bioinformatic analysis of the published single-cell RNA-seq data collected from AD patient cohorts indicates that the Trpc3 gene is uniquely upregulated in excitatory neurons. TRPC3 expression is also upregulated in post-mortem AD brains, and in both acute and chronic mouse models of AD. Functional screening of TRPC3 antagonists resulted in a lead inhibitor JW-65, which completely rescued Aβ-induced neurotoxicity, impaired synaptic plasticity (e.g., LTP), and learning memory in acute and chronic experimental AD models. In cultured rat hippocampal neurons, we found that treatment with soluble β-amyloid oligomers (AβOs) induces rapid and sustained upregulation of the TRPC3 expression selectively in excitatory neurons. This aberrantly upregulated TRPC3 contributes to AβOs-induced Ca 2+ overload through the calcium entry and store-release mechanisms. The neuroprotective action of JW-65 is primarily mediated via restoring AβOs-impaired Ca 2+ /calmodulin-mediated signaling pathways, including calmodulin kinases CaMKII/IV and calcineurin (CaN). The synaptic protective mechanism via TRPC3 inhibition was further supported by hippocampal RNA-seq data from the symptomatic 5xFAD mice after chronic treatment with JW-65. Overall, these findings not only validate TRPC3 as a novel therapeutic target for treating synaptic dysfunction of AD but most importantly, disclose a distinct role of upregulated TRPC3 in AD pathogenesis in mediating Ca 2+ dyshomeostasis.

CaMKK2: bridging the gap between Ca2+ signaling and energy-sensing.

Calcium (Ca2+) ions are ubiquitous and indispensable signaling messengers that regulate virtually every cell function. The unique ability of Ca2+ to regulate so many different processes yet cause stimulus specific changes in cell function requires sensing and decoding of Ca2+ signals. Ca2+-sensing proteins, such as calmodulin, decode Ca2+ signals by binding and modifying the function of a diverse range of effector proteins. These effectors include the Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) enzyme, which is the core component of a signaling cascade that plays a key role in important physiological and pathophysiological processes, including brain function and cancer. In addition to its role as a Ca2+ signal decoder, CaMKK2 also serves as an important junction point that connects Ca2+ signaling with energy metabolism. By activating the metabolic regulator AMP-activated protein kinase (AMPK), CaMKK2 integrates Ca2+ signals with cellular energy status, enabling the synchronization of cellular activities regulated by Ca2+ with energy availability. Here, we review the structure, regulation, and function of CaMKK2 and discuss its potential as a treatment target for neurological disorders, metabolic disease, and cancer.

CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

Protein quality control serves as the primary defense mechanism for cells against proteotoxicity induced by proteasome dysfunction. While cells can limit the build-up of ubiquitinated misfolded proteins during proteasome inhibition, the precise mechanism is unclear. Here, we find that protein kinase Ca2+/Calmodulin (CaM)-dependent protein kinase II (CaMKII) maintains proteostasis during proteasome inhibition. We show that proteasome inhibition activates CaMKII, which phosphorylates B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) at residues S173, S377, and S386. Phosphorylated BAG3 activates the heme-regulated inhibitor (HRI)- eukaryotic initiation factor-2α (eIF2α) signaling pathway, suppressing protein synthesis and the production of aggregated ubiquitinated misfolded proteins, ultimately mitigating the proteotoxic crisis. Inhibition of CaMKII exacerbates the accumulation of aggregated misfolded proteins and paraptosis induced by proteasome inhibitors. Based on these findings, we validate that combined targeting of proteasome and CaMKII accelerates tumor cell death and enhances the efficacy of proteasome inhibitors in tumor treatment. Our data unveil a new proteasomal inhibition-induced misfolded protein quality control mechanism and propose a novel therapeutic intervention for proteasome inhibitor-mediated tumor treatment.

IRS2 Signaling Protects Against Stress-Induced Arrhythmia by Maintaining Ca2+ Homeostasis.

The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of IRS2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease. A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice. The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²+ transient amplitudes, increased spontaneous Ca²+ sparks, and reduced sarcoplasmic reticulum Ca²+ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca2+/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice. Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

TAK-242 alleviates diabetic cardiomyopathy via inhibiting pyroptosis and TLR4/CaMKII/NLRP3 pathway.

Diabetic cardiomyopathy (DCM) is identified as a progressive disease that may lead to irreparable heart failure. Toll-like receptor (TLR) signaling is believed to be implicated in the pathogenesis of DCM. This study intended to explore the potential impact of Toll-like receptor 4 (TLR4) on DCM in vitro and in vivo. Streptozotocin and HG medium were utilized to induce diabetes in animal and cell models, respectively. Selective TLR4 inhibitor TAK-242 and calcium/calmodulin-dependent protein kinase-II (CaMKII) inhibitor KN-93 were employed to explore the involvement of TLR4/CaMKII in DCM. TLR4 expression was increased in DCM hearts, while inhibition of TLR4 activation by TAK-242 improved cardiac function, attenuated heart hypertrophy, and fibrosis, as well as reduced oxidative stress and proinflammatory cytokine levels in rats, which were confirmed by Doppler echocardiography, hematoxylin and eosin staining, and Masson Trichome staining and specific enzyme-linked immunosorbent assay kits. Besides, the expression of hypertrophy-related molecules and oxidative stress damage were also inhibited by TAK-242. Furthermore, TAK-242 treatment reduced CaMKII phosphorylation accompanied by decreased expression of NOD-like pyrin domain-containing protein 3, gasdermin D (GSDMD), The N-terminal domain of Gasdermin D (GSDMD-N), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and Caspase-1 both in vivo and in vitro. Similar positive impacts on HG-induced pyroptosis were also observed with KN-93 treatment, and this was achieved without affecting TLR4 expression. Collectively, our work suggested that TAK-242 demonstrated substantial benefits against DCM both in vivo and in vitro, potentially attributed to the suppression of the TLR4-mediated CaMKII/NLRP3 pathway activity.

Variants in LRRC7 lead to intellectual disability, autism, aggression and abnormal eating behaviors

Members of the leucine rich repeat (LRR) and PDZ domain (LAP) protein family are essential for animal development and histogenesis. Densin-180, encoded by LRRC7, is the only LAP protein selectively expressed in neurons. Densin-180 is a postsynaptic scaffold at glutamatergic synapses, linking cytoskeletal elements with signalling proteins such as the α-subunit of Ca2+/calmodulin-dependent protein kinase II. We have previously observed an association between high impact variants in LRRC7 and Intellectual Disability; also three individual cases with variants in LRRC7 had been described. We identify here 33 individuals (one of them previously described) with a dominant neurodevelopmental disorder due to heterozygous missense or loss-of-function variants in LRRC7. The clinical spectrum involves intellectual disability, autism, ADHD, aggression and, in several cases, hyperphagia-associated obesity. A PDZ domain variant interferes with synaptic targeting of Densin-180 in primary cultured neurons. Using in vitro systems (two hybrid, BioID, coimmunoprecipitation of tagged proteins from 293T cells) we identified new candidate interaction partners for the LRR domain, including protein phosphatase 1 (PP1), and observed that variants in the LRR reduced binding to these proteins. We conclude that LRRC7 encodes a major determinant of intellectual development and behaviour.

An L-type calcium channel blocker nimodipine exerts anti-fibrotic effects by attenuating TGF-β1 induced calcium response in an in vitro model of thyroid eye disease

BackgroundThyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-β1 induced in vitro TED model.MethodsPrimary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca2+ response on TGF-β1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-β1 induced in vitro TED model. The roles of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways.ResultsLTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca2+ response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway.ConclusionsTGF-β1 induces an LTCC-mediated Ca2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED.

Precision therapy targeting CAMK2 to overcome resistance to EGFR inhibitors in FAT1-mutated oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a prevalent type of cancer with a high mortality rate in its late stages. One of the major challenges in OSCC treatment is the resistance to epidermal growth factor receptor (EGFR) inhibitors. Therefore, it is imperative to elucidate the mechanism underlying drug resistance and develop appropriate precision therapy strategies to enhance clinical efficacy. To evaluate the efficacy of the combination of the Ca2+/calmodulin-dependent protein kinase II (CAMK2) inhibitor KN93 and EGFR inhibitors, we performed in vitro and in vivo experiments using two FAT atypical cadherin 1 (FAT1)-deficient (SCC9 and SCC25) and two FAT1 wild-type (SCC47 and HN12) OSCC cell lines. We assessed the effects of EGFR inhibitors (afatinib or cetuximab), KN93, or their combination on the malignant phenotype of OSCC in vivo and in vitro. The alterations in protein expression levels of members of the EGFR signaling pathway and SRY-box transcription factor 2 (SOX2) were analyzed. Changes in the yes-associated protein 1 (YAP1) protein were characterized. Moreover, we analyzed mitochondrial dysfunction. Besides, the effects of combination therapy on mitochondrial dynamics were also evaluated. OSCC with FAT1 mutations exhibited resistance to EGFR inhibitors treatment. The combination of KN93 and EGFR inhibitors significantly inhibited the proliferation, survival, and migration of FAT1-mutated OSCC cells and suppressed tumor growth in vivo. Mechanistically, combination therapy enhanced the therapeutic sensitivity of FAT1-mutated OSCC cells to EGFR inhibitors by modulating the EGFR pathway and downregulated tumor stemness-related proteins. Furthermore, combination therapy induced reactive oxygen species (ROS)-mediated mitochondrial dysfunction and disrupted mitochondrial dynamics, ultimately resulting in tumor suppression. Combination therapy with EGFR inhibitors and KN93 could be a novel precision therapeutic strategy and a potential clinical solution for EGFR-resistant OSCC patients with FAT1 mutations.

CPK10 regulates low light-induced tomato flower drop downstream of IDL6 in a calcium-dependent manner.

Flower drop is a major cause for yield loss in many crops. Previously, we found that the tomato (Solanum lycopersicum) INFLORESCENCE DEFICIENT IN ABSCISSION-Like (SlIDL6) gene contributes to flower drop induced by low light. However, the molecular mechanisms by which SlIDL6 acts as a signal to regulate low light-induced abscission remain unclear. In this study, SlIDL6 was found to elevate cytosolic Ca2+ concentrations ([Ca2+]cyt) in the abscission zone (AZ), which was required for SlIDL6-induced flower drop under low light. We further identified that 1 calcium-dependent protein kinase gene, SlCPK10, was highly expressed in the AZ and upregulated by SlIDL6-triggered [Ca2+]cyt. Overexpression and knockout of SlCPK10 in tomato resulted in accelerated and delayed abscission, respectively. Genetic evidence further indicated that knockout of SlCPK10 significantly impaired the function of SlIDL6 in accelerating abscission. Furthermore, Ser-371 phosphorylation in SlCPK10 dependent on SlIDL6 was necessary and sufficient for its function in regulating flower drop, probably by stabilizing the SlCPK10 proteins. Taken together, our findings reveal that SlCPK10, as a downstream component of the IDL6 signaling pathway, regulates flower drop in tomato under low-light stress.

Multi-faceted potential of sophoridine compound's anti-arrhythmic and antioxidant effects through ROS/CaMKII pathway

Cardiac arrhythmias remain a significant cause of mortality and morbidity, for novel antiarrhythmic therapies. This study states that the first report of sophoridine (SPN), a quinolizidine alkaloid derived from traditional Chinese herbs, shows promise as a potential candidate due to its anti-arrhythmic and antioxidant properties. The study found that cell viability in H9C2 rat cardiomyocytes remained stable even when treated with SPN at a higher dosage of 100 μg/ml. This phenomenon was accompanied by increases in mitochondria-derived reactive oxygen species (ROS) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling, at 50 and 100 μg/ml. Glucose fluctuations regulate ventricular arrhythmias caused by SPN by activating the ROS/CaMKII pathway. Experimental models using zebrafish provided additional evidence supporting the regulatory effects of SPN on heart rate. In addition, the administration of SPN resulted in substantial deregulation of crucial genes involved in heart development (nppa, nppb, tnnt2a) at the transcriptional level in zebrafish. These findings provide insight into the various pharmacological properties of SPN and this opens up new possibilities for anti-arrhythmic treatment strategies.

Employing Hexahydroquinolines as PfCDPK4 Inhibitors to Combat Malaria Transmission: An Advanced Computational Approach.

Existing antimalarial drugs primarily target blood-stage parasites, but there is a need for transmission-blocking drugs to combat malaria effectively. Plasmodium falciparum Calcium-dependent Protein Kinase 4 (CDPK4) is a promising target for such drugs. This study employed advanced in silico analyses of hexahydroquinolines (HHQ) derivatives to identify PfCDPK4 inhibitors capable of disrupting malaria transmission. Structure-based virtual screening (SBVS) was employed to discover HHQ derivatives with the highest binding affinities against the 3D structure of PfCDPK4 (PDB 1D: 4QOX). Interaction analysis of protein-ligand complexes utilized Discovery Studio Client, while druglikeness and ADMET properties were assessed using SwissADME and pkCSM web servers, respectively. Quantum mechanical calculations of the top hits were conducted using density functional theory (DFT), and GROMACS was employed to perform the molecular dynamics (MD) simulations. Binding free energy was predicted using the MMPBSA.py tool from the AMBER package. SBVS identified ten best hits possessing docking scores within the range of -11.2 kcal/mol and -10.6 kcal/mol, surpassing the known inhibitor, BKI-1294 (-9.9 kcal/mol). Among these, 4-[4-(Furan-2-carbonyl)piperazin-1-yl]-1-(naphthalen-2-ylmethyl)-2-oxo-4a,5,6,7,8,8a-hexahydroquinoline-3-carbonitrile (PubChem ID: 145784778) exhibited the highest binding affinity (-11.2 kcal/mol) against PfCDPK4. Comparative analysis of this compound with BKI-1294 using advanced computational approaches demonstrated competitive potential. These findings suggest the potential of 4-[4-(Furan-2-carbonyl)piperazin-1-yl]-1-(naphthalen-2-ylmethyl)-2-oxo-4a,5,6,7,8,8a-hexahydroquinoline-3-carbonitrile as a promising PfCDPK4 inhibitor for disrupting malaria transmission. However, further experimental studies are warranted to validate its efficacy and safety profile.

Phosphorylation of PIP2;7 by CPK28 or Phytophthora kinase effectors dampens pattern-triggered immunity in Arabidopsis

Plasma membrane intrinsic proteins (PIPs), a subclass of aquaporins, play an important role in plant immunity by acting as H2O2 transporters. Their homeostasis is mostly maintained by C-terminal serine phosphorylation. However, the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown. Here, we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H2O2. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation. During pathogen infection, CPK28 dissociates from PIP2;7 and destabilizes, leading to PIP2;7 accumulation. As a countermeasure, oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation. Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28. Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection. Accordingly, we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant. The strategy only allows accumulation of PIP2;7S273/276A during infection to limit potential side effects on normal plant growth.

ZmCRK1 negatively regulates maize's response to drought stress by phosphorylating plasma membrane H+-ATPase ZmMHA2.

Drought severely affects crop growth and yields. Stomatal regulation plays an important role in plant response to drought stress. Light-activated plasma membrane-localized proton ATPase (PM H+-ATPase) mainly promoted the stomatal opening. Abscisic acid (ABA) plays a dominant role in the stomatal closure during drought stress. It is not clear how PM H+-ATPase is involved in the regulation of ABA-induced stomatal closure. We found that a CALCIUM-DEPENDENT PROTEIN KINASE RELATED KINASE 1 (ZmCRK1), and its mutant zmcrk1 exhibited slow water loss in detached leaves, high-survival rate after drought stress, and sensitivity to stomatal closure induced by ABA. The ZmCRK1 overexpression lines are opposite. ZmCRK1 interacted with the maize PM H+-ATPase ZmMHA2. ZmCRK1 phosphorylated ZmMHA2 at the Ser-901 and inhibited its proton pump activity. ZmCRK1 overexpression lines and zmmha2 mutants had low H+-ATPase activity, resulting in impaired ABA-induced H+ efflux. Taken together, our study indicates that ZmCRK1 negatively regulates maize drought stress response by inhibiting the activity of ZmMHA2. Reducing the expression level of ZmCRK1 has the potential to reduce yield losses under water deficiency.

LT-102, an AMPA receptor potentiator, alleviates depression-like behavior and synaptic plasticity impairments in prefrontal cortex induced by sleep deprivation

BackgroundSleep loss is closely related to the onset and development of depression, and the mechanisms involved may include impaired synaptic plasticity. Considering the important role of glutamate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) in synaptic plasticity as well as depression, we introduce LT-102, a novel AMPARs potentiator, to evaluate the potential of LT-102 in treating sleep deprivation-induced depression-like behaviors. MethodsWe conducted a comprehensive behavioral assessment to evaluate the effects of LT-102 on depression-like symptoms in male C57BL/6J mice. This assessment included the open field test to measure general locomotor activity and anxiety-like behavior, the forced swimming test and tail suspension test to assess despair behaviors indicative of depressive states, and the sucrose preference test to quantify anhedonia, a core symptom of depression. Furthermore, to explore the impact of LT-102 on synaptic plasticity, we utilized a combination of Western blot analysis to detect protein expression levels, Golgi-Cox staining to visualize neuronal morphology, and immunofluorescence to examine the localization of synaptic proteins. Additionally, we utilized primary cortical neurons to delineate the signaling pathway modulated by LT-102. ResultsTreatment with LT-102 significantly reduced depression-like behaviors associated with sleep deprivation. Quantitative Western blot (WB) analysis revealed a significant increase in GluA1 phosphorylation in the prefrontal cortex (PFC), triggering the Ca2+/calmodulin-dependent protein kinase II/cAMP response element-binding protein/brain-derived neurotrophic factor (CaMKII/CREB/BDNF) and forkhead box protein P2/postsynaptic density protein 95 (FoxP2/PSD95) signaling pathways. Immunofluorescence imaging confirmed that LT-102 treatment increased spine density and co-labeling of PSD95 and vesicular glutamate transporter 1 (VGLUT1) in the PFC, reversing the reductions typically observed following sleep deprivation. Golgi staining further validated these results, showing a substantial increase in neuronal dendritic spine density in sleep-deprived mice treated with LT-102. Mechanistically, application of LT-102 to primary cortical neurons, resulted in elevated levels of phosphorylated AKT (p-AKT) and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β), key downstream molecules in the BDNF signaling pathway, which in turn upregulated FoxP2 and PSD95 expression. LimitationsIn our study, we chose to exclusively use male mice to eliminate potential influences of the estrous cycle on behavior and physiology. As there is no widely accepted positive drug control for sleep deprivation studies, we did not include one in our research. ConclusionOur results suggest that LT-102 is a promising therapeutic agent for counteracting depression-like behaviors and synaptic plasticity deficits induced by sleep deprivation, primarily through the activation of CaMKII/CREB/BDNF and AKT/GSK3β/FoxP2/PSD95 signaling pathways.

SlCPK27 cross-links SlHY5 and SlPIF4 in brassinosteroid-dependent photo- and thermo-morphogenesis in tomato.

ELONGATED HYPOCOTOYL5 (HY5) and PHYTOCHROME INTERACTING FACTORs (PIFs) are two types of important light-related regulators of plant growth, however, their interplay remains elusive. Here, we report that the activated tomato (Solanum lycopersicum) HY5 (SlHY5) triggers the transcription of a Calcium-dependent Protein Kinase SlCPK27. SlCPK27 interacts with and phosphorylates SlPIF4 at Ser-252 and Ser-308 phosphosites to promote its degradation. SlPIF4 promotes hypocotyl elongation mainly by activating the transcription of SlDWF, a key gene in brassinosteroid (BR) biosynthesis. Such a SlHY5-SlCPK27-SlPIF4-BR cascade not only plays a crucial role in photomorphogenesis but also regulates thermomorphogenesis. Our results uncover a previously unidentified mechanism that integrates Ca2+ signaling with the light signaling pathways to regulate plant growth by modulating BR biosynthesis in response to changes in ambient light and temperature.

CaMKII protein expression and phosphorylation in human skeletal muscle by immunoblotting: Isoform specificity

Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) is activated during exercise by reactive oxygen species (ROS) and Ca2+ transients initiating muscle contraction. CaMKII modulates antioxidant, inflammatory, metabolic and autophagy signalling pathways. CaMKII is coded by four homologous genes (α, β, γ, and δ). In rat skeletal muscle, δD, δA, γD, γB and βM have been described while different characterisations of human skeletal muscle CaMKII isoforms have been documented. Precisely discerning between the various isoforms is pivotal for understanding their distinctive functions and regulatory mechanisms in response to exercise and other stimuli. This study aimed to optimize the detection of the different CaMKII isoforms by western blotting using eight different CaMKII commercial antibodies in human skeletal muscle. Exercise-induced posttranslational modifications, i.e. phosphorylation and oxidations, allowed the identification of specific bands by multitargeting them with different antibodies after stripping and reprobing. The methodology proposed has confirmed the molecular weight of βM CaMKII and allows distinguishing between γ/δ and δD CaMKII isoforms. The corresponding molecular weight for the CaMKII isoforms resolved were: δD, at 54.2 ± 2.1 kDa; γ/δ, at 59.0 ± 1.2 kDa and 61.6 ± 1.3 kDa; and βM isoform, at 76.0 ± 1.8 kDa. Some tested antibodies showed high specificity for the δD, the most responsive isoform to ROS and intracellular Ca2+ transients in human skeletal muscle, while others, despite the commercial claims, failed to show such specificity.

The phosphorylation of Smad3 by CaMKIIγ leads to the hepatocyte pyroptosis under perfluorooctane sulfonate exposure

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and accumulated in the liver of mammals. PFOS exposure is closely associated with the development of pyroptosis. Nevertheless, the underlying mechanism is unclear. We found here that PFOS induced pyroptosis in the mice liver and L-02 cells as demonstrated by activation of the NOD-like receptor protein 3 inflammasome, gasdermin D cleavage and increased release of interleukin-1β and interleukin-18. The level of cytoplasmic calcium was accelerated in hepatocytes upon exposure to PFOS. The phosphorylated/activated form of calcium/calmodulin-dependent protein kinase II (CaMKII) was augmented by PFOS in vivo and in vitro. PFOS-induced pyroptosis was relieved by CaMKII inhibitor. Among various CaMKII subtypes, we identified that CaMKIIγ was activated specifically by PFOS. CaMKIIγ interacted with Smad family member 3 (Smad3) under PFOS exposure. PFOS increased the phosphorylation of Smad3, and CaMKII inhibitor or CaMKIIγ siRNA alleviated PFOS-caused phosphorylation of Smad3. Inhibiting Smad3 activity was found to alleviate PFOS-induced hepatocyte pyroptosis. This study puts forward that CaMKIIγ-Smad3 is the linkage between calcium homeostasis disturbance and pyroptosis, providing a mechanistic explanation for PFOS-induced pyroptosis.