Calmodulin-dependent Kinase Research Articles

The pivotal role of the Hes1/Piezo1 pathway in the pathophysiology of glucocorticoid-induced osteoporosis.

Glucocorticoid-induced osteoporosis (GIOP) lacks fully effective treatments. This study investigated the role of Piezo1, a mechanosensitive ion channel component 1, in GIOP. We found reduced Piezo1 expression in cortical bone osteocytes from patients with GIOP and a GIOP mouse model. Yoda1, a Piezo1 agonist, enhanced the mechanical stress response and bone mass and strength, which were diminished by dexamethasone (DEX) administration in GIOP mice. RNA-seq revealed that Yoda1 elevated Piezo1 expression by activating the key transcription factor Hes1, followed by enhanced CaM kinase II and Akt phosphorylation in osteocytes. This improved the lacuno-canalicular network and reduced sclerostin production and the receptor activator of NF-κB/osteoprotegerin ratio, which were mitigated by DEX. Comparative analysis of mouse models and human GIOP cortical bone revealed downregulation of mechanostimulated osteogenic factors, such as osteocrin, and cartilage differentiation markers in osteoprogenitor cells. In human periosteum-derived cells, DEX suppressed differentiation into osteoblasts, but Yoda1 rescued this effect. Our findings suggest that reduced Piezo1 expression and activity in osteocytes and periosteal cells contribute to GIOP, and Yoda1 may offer a novel therapeutic approach by restoring mechanosensitivity.

Endogenous hydrogen peroxide positively regulates secretion of a gut-derived peptide in neuroendocrine potentiation of the oxidative stress response in Caenorhabditis elegans.

The gut-brain axis mediates bidirectional signaling between the intestine and the nervous system and is critical for organism-wide homeostasis. Here, we report the identification of a peptidergic endocrine circuit in which bidirectional signaling between neurons and the intestine potentiates the activation of the antioxidant response in Caenorhabditis elegans in the intestine. We identify an FMRF-amide-like peptide, FLP-2, whose release from the intestine is necessary and sufficient to activate the intestinal oxidative stress response by promoting the release of the antioxidant FLP-1 neuropeptide from neurons. FLP-2 secretion from the intestine is positively regulated by endogenous hydrogen peroxide (H2O2) produced in the mitochondrial matrix by sod-3/superoxide dismutase, and is negatively regulated by prdx-2/peroxiredoxin, which depletes H2O2 in both the mitochondria and cytosol. H2O2 promotes FLP-2 secretion through the DAG and calcium-dependent protein kinase C family member pkc-2 and by the SNAP25 family member aex-4 in the intestine. Together, our data demonstrate a role for intestinal H2O2 in promoting inter-tissue antioxidant signaling through regulated neuropeptide-like protein exocytosis in a gut-brain axis to activate the oxidative stress response.

Mitochondrial metabolic reprogramming of macrophages and T cells enhances CD47 antibody-engineered oncolytic virus antitumor immunity

BackgroundAlthough immunotherapy can reinvigorate immune cells to clear tumors, the response rates are poor in some patients. Here, CD47 antibody-engineered oncolytic viruses (oAd-αCD47) were employed to lyse tumors and activate...

CaMKII-dependent non-canonical RIG-I pathway promotes influenza virus propagation in the acute-phase of infection.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is one of hundreds of host-cell factors involved in the propagation of type A influenza virus (IAV), although its mechanism of action is unknown. Here, we identified CaMKII inhibitory peptide M3 by targeting its kinase domain using affinity-based screening of a tailored random peptide library. M3 inhibited IAV cytopathicity and propagation in cells by specifically inhibiting the acute-phase activation of retinoic acid-inducible gene I (RIG-I), which is uniquely regulated by CaMKII. Downstream of the RIG-I pathway activated TBK1 and then IRF3, which induced small but sufficient amounts of transcripts of the genes for IFN α/β to provide the capped 5'-ends that were used preferentially as primers to synthesize viral mRNAs by the cap-snatching mechanism. Importantly, knockout of RIG-I in cells almost completely inhibited the expression of IFN mRNAs and subsequent viral NP mRNA early in infection (up to 6 h after infection), which then protected cells from cytopathicity 24 h after infection. Thus, CaMKII-dependent acute-phase activation of RIG-I promoted IAV propagation, whereas the canonical RIG-I pathway stimulated antiviral activity by inducing large amounts of mRNA for IFNs and then for antiviral proteins later in infection. Co-administration of M3 with IAV infection rescued mice from the lethality and greatly reduced proinflammatory cytokine mRNA expression in the lung, indicating that M3 is highly effective against IAV in vivo. Thus, regulation of the CaMKII-dependent non-canonical RIG-I pathway may provide a novel host-factor-directed antiviral therapy.IMPORTANCEThe recent emergence of IAV strains resistant to commonly used therapeutic agents that target viral proteins has exacerbated the need for innovative strategies. Here, we originally identified CaMKII-inhibitory peptide M3, which efficiently inhibits IAV-lethality in vitro and in vivo. M3 specifically inhibited the acute-phase activation of RIG-I, which is a novel pathway to promote IAV propagation. Thus, this pathway acts in an opposite manner compared with the canonical RIG-I pathway, which plays essential roles in antiviral innate immune response later in infection. The CaMKII-dependent non-canonical RIG-I pathway can be a promising and novel drug target for the treatment of infections.

Goose astrovirus induces apoptosis and endoplasmic reticulum stress in gosling hepatocytes

The ongoing Goose astrovirus (GoAstV) epidemic, which primarily infects goslings causing severe liver damage, has inflicted considerable damage on the poultry industry. Endoplasmic reticulum stress (ERS) is a significant modulator of several viral infections, while severe ERS may result in apoptosis. This study examined the roles and possible mechanisms of ERS and apoptosis in GoAstV-induced liver injury in goslings. Two hundred Xingguo gray geese were chosen and randomly separated into two groups (Con and Dis). The Dis group received a subcutaneous injection of GoAstV genotype 2 (GoAstV-2) JX01 (2 × 106 TCID50/0.2 mL), whereas the Con group received a subcutaneous injection of 0.2 mL physiological saline, both at 1 day of life. Subsequent analyses demonstrate that the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased following GoAstV infection. Hematoxylin and eosin (HE) staining revealed swollen and ruptured hepatocytes, with significant inflammatory cell infiltration. Electron microscopy revealed expansion of the endoplasmic reticulum (ER) and aggregation of chromatin at the periphery. TUNEL testing further demonstrated an increase in the quantity of positive cells. RT-qPCR and Western blot analyses indicated that GoAstV infection enhanced the expression of ER Ca2+ release channels (IP3R and RYR) and calmodulin-dependent protein kinase II (CaMKII), while decreasing the expression of ER Ca2+ uptake channels (SERCA). Further, GoAstV infection activated ERS-related factors, including GRP78, IRE1α, PERK, ATF6, eIF2α, ATF4, CHOP, TRAF2, and JNK, induced the expression of pro-apoptotic factors (Caspase-3, Caspase-9, and Bax), and inhibited the mRNA and protein expression of the anti-apoptotic factor Bcl-2. Correlation analysis further revealed a potential relationship among ERS gene expression, apoptotic gene expression, and liver injury. In summary, GoAstV infection can lead to liver injury by interfering with ER Ca2+ homeostasis, exacerbating ERS and inducing hepatocyte apoptosis.

Overexpression of StCDPK13 in Potato Enhances Tolerance to Drought Stress

Calcium-dependent protein kinases (CDPKs), which are activated by transient changes in the Ca2+ concentration in plants, are important for various biological processes, such as growth, development, defense against biotic and abiotic stresses, and others. Mannitol is commonly used as an osmotic regulatory substance in culture medium or nutrient solutions to create water-deficit conditions. Here, we cloned the potato (Solanum tuberosum L.) StCDPK13 gene and generated stable transgenic StCDPK13-overexpression potato plants. To investigate the potential functions of StCDPK13 in response to drought stress, overexpression-transgenic (OE1, OE2, and OE7) and wild-type (WT) potato seedlings were cultured on MS solid media without or with mannitol, representing the control or drought stress, for 20 days; the elevated mannitol concentrations (150 and 200 mM) were the drought stress conditions. The StCDPK13 gene was consistently expressed in different tissues and was induced by drought stress in both OE and WT plants. The phenotypic traits and an analysis of physiological indicators revealed that the transgenic plants exhibited more tolerance to drought stress than the WT plants. The overexpression lines showed an increased plant height, number of leaves, dry shoot weight, root length, root number, root volume, number of root tips, fresh root weight, and dry root weight under drought stress. In addition, the activities of antioxidant enzymes (CAT, SOD, and POD) and the accumulation of proline and neutral sugars were significantly increased, whereas the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and O2•−, were significantly reduced in the OE lines compared to WT plants under drought stress. Moreover, the stomatal aperture of the leaves and the water loss rate in the leaves of the OE lines were significantly reduced under drought stress compared to the WT plants. In addition, the overexpression of StCDPK13 upregulated the expression levels of stress-related genes under drought stress. Collectively, these results indicate that the StCDPK13 gene plays a positive role in drought tolerance by reducing the stomatal aperture, promoting ROS scavenging, and alleviating oxidative damage under drought stress in potatoes.

Differential participation of CaMKII/ROCK and NOS pathways in the cholinergic inhibitory drive operated by nicotinic α7 receptors in perisynaptic Schwann cells

Nicotinic α7 receptors (α7 nAChRs) present in perisynaptic Schwann cells (PSCs) control acetylcholine (ACh) spillover from the neuromuscular synapse by transiently increasing intracellular Ca2+, which fosters adenosine release via type 1 equilibrative nucleoside transporters (ENT1) and retrograde activation of presynaptic A1 inhibitory receptors. The putative Ca2+-dependent pathways downstream α7 nAChRs involved in the sensing inhibitory drive operated by PSCs is unknown. Herein, we used phrenic nerve-hemidiaphragm preparations from Wistar rats. Time-lapse video-microscopy was instrumental to assess nerve-evoked (50-Hz bursts) transmitter exocytosis and intracellular NO oscillations in nerve terminals and PSCs loaded with FM4-64 and DAF-FM diacetate fluorescent dyes, respectively. Selective activation of α7 nAChRs with PNU 282987 reduced transmitter exocytosis (FM4-64 dye unloading) during 50-Hz bursts. Inhibition of calmodulin activity (with W-7), Ca2+/calmodulin-dependent protein kinase II (CaMKII; with KN-62) and Rho-kinase (ROCK; with H1152) all prevented the release inhibitory effect of PNU 282987. The α7 nAChR agonist transiently increased NO inside PSCs; the same occurred during phrenic nerve stimulation with 50-Hz bursts in the presence of the cholinesterase inhibitor, neostigmine. The nitric oxide synthase (NOS) inhibitor, L-NOARG, but not with the guanylylcyclase (GC) inhibitor, ODQ, prevented inhibition of transmitter exocytosis by PNU 282987. Inhibition of adenosine kinase with ABT 702 favors the intracellular accumulation and translocation of the nucleoside to the synaptic cleft, thus overcoming prevention of the PNU 282987 effect caused by H1152, but not by L-NOARG. In conclusion, the α7nAChR-mediated cholinergic inhibitory drive operated by PSCs involves two distinct Ca2+-dependent intracellular pathways: a CaMKII/ROCK cascade along with a GC-independent NO pathway with divergent end-effects concerning ADK inhibition.

Selective inhibition of T-type calcium channel preserves ischemic pre-conditioning mediated neuroprotection during cerebral ischemia reperfusion injury in diabetic mice.

Ischemic preconditioning (IPC) provides ischemic tolerance and neuroprotection during cerebral ischemia reperfusion (CI/R) injury. Diabetes abolishes the beneficial effects of conditioning phenomenon during CI/R. The study investigates the role of T-type calcium ion channel in IPC mediated protection during diabetes mellitus. The study employed Swiss Albino mice. Animals were divided into 3 normoglycaemic groups (Sham, CI/R, and IPC) and 4 hyperglycaemic groups (Sham, CI/R, IPC, and ML218 + IPC). CI/R injury was induced in Swiss Albino mice by occlusion of common carotid arteries followed by reperfusion. IPC was given prior to CI/R injury and diabetes was induced using streptozotocin (STZ). Animals were assessed for learning, memory, motor coordination, neurological function, cerebral infarction, edema, and histopathological alterations. Biochemical assessments were performed for calcium binding proteins (Calmodulin (CaM), calcium/calmodulin-dependent protein kinase II (CaMKII), and S100B), oxidative stress (4-hydroxy-2-nonenal (4-HNE)), glutathione (GSH), inflammation (nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), tumor necrosis factor (TNF-α), interleukin (IL-10)), inducible nitric oxide synthase (iNOS) levels, and acetylcholinesterase activity (AChE) in brain supernatants. NF-kB, iNOS, and S100B serum levels were also assessed. CI/R animals (normoglycemic and hyperglycaemic) showed impairment in learning, memory, motor coordination, and neurological function along with increase in cerebral infarction, edema, and histopathological alterations. Furthermore, increase in brain calcium-binding proteins, oxidative stress, inflammation, and AChE along with serum NF-kB, iNOS, and S100B levels were recorded in CI/R animals. IPC ameliorated CI/R induced behavioral, biochemical, and histopathological impairment, however no beneficial effects were observed in IPC (diabetic) mice. Administration of ML218 (10mg/kg; i.p.), a selective T-type calcium channel re-established the IPC mediated neuroprotection in CI/R diabetic animals. In conclusion, IPC-mediated neuroprotection was abolished in diabetic mice. T-type calcium ion channel antagonism plays an important role in the IPC-mediated neuroprotection during hyperglycaemia.

Genomic survey and evolution analysis of calcium-dependent protein kinases in plants and their stress-responsive patterns in populus

BackgroundCalcium-dependent protein kinases (CDPKs) phosphorylate downstream target proteins in response to signals transmitted by free calcium ions (Ca2+, one of the second messengers) and thus play important regulatory roles in many biological processes, such as plant growth, development, and stress response.ResultsA bioinformatic analysis, as well as thorough evolutionary and expression investigations, were conducted to confirm previous reports of functional evidence for plant CDPKs. Using the Phytozome database’s BLAST search engine and the HMM search tool in TBtools software, we discovered that CDPKs are well conserved from green algae to flowering angiosperms in various gene family sizes. Additional investigations of the obtained CDPKs revealed high conservation of domain and motif numbers, gene architectures, and patterns. However, this conservation differed among plant species. Phylogenetic analysis demonstrated that the CDPK gene family diverged from a common ancient gene. Similarly, investigations into plant interspecies evolutionary relationships revealed common ancestral plant species, suggesting speciation of plants and evolution based on plant adaptation and diversification. A search for the driving force of CDPK gene family expansion revealed that dispersed duplication events, among other duplication events, contributed largely to CDPK gene family expansion. Gene localization analysis in P. trichocarpa demonstrated that most CDPK genes are localized within several cell organelles and bind other kinases and proteins to perform their biological functions efficiently. Using RNA-seq data and qPCR analyses, we postulated that PtCDPKs play functional roles in abiotic stress responses by regulating cold, heat, drought and salt stress to varying extents.ConclusionThe CDPK genes are well conserved in plants and are critical entities in abiotic stress regulation, and further exploration and manipulation of these genes in the future may provide solutions to some of the challenges in agriculture, forestry and food security.

A revised view of the role of CaMKII in learning and memory.

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) plays a fundamental role in learning and possibly also in memory. However, current mechanistic models require fundamental revision. CaMKII autophosphorylation at Thr286 (pThr286) does not provide the molecular basis for long-term memory, as long believed. Instead, pThr286 mediates the signal processing required for induction of several distinct forms of synaptic plasticity, including Hebbian long-term potentiation and depression and non-Hebbian behavioral timescale synaptic plasticity. We discuss (i) the molecular computations by which CaMKII supports these diverse plasticity mechanisms, (ii) alternative CaMKII mechanisms that may contribute to the maintenance phase of LTP and (iii) the relationship of these mechanisms to behavioral learning and memory.

Immunofluorescent Localization of Ca²⁺-Sensor Proteins in The Somatic Motor Muscles of The Earthworm <i>Lumbricus terrestris</i>

The method of immunofluorescent staining of earthworm somatic muscle samples showed the presence of calmodulin, Ca²⁺-calmodulin-dependent protein kinases type 1 and type 2, synaptotagmin type 2 and type 7 and calcineurin A. These proteins are detected in both synaptic and extrasynaptic regions of the motor muscle. However, for synaptotagmin type 2 and type 7, calcineurin A, their predominant localization in the area of neuromuscular synapses has been established. Besides, synaptic localization for synaptotagmin 7 and calcineurin A is most clearly expressed.

RETRACTION: Spike Protein of SARS-CoV Stimulates Cyclooxygenase-2 Expression via Both Calcium-Dependent and Calcium-Independent Protein Kinase C Pathways.

M. Liu, Y. Yang, C. Gu, Y. Yue, K. K. Wu, J. Wu, and Y. Zhu, "Spike Protein of SARS-CoV Stimulates Cyclooxygenase-2 Expression via Both Calcium-Dependent and Calcium-Independent Protein Kinase C Pathways," The FASEB Journal 21, no. 7 (2007): 1586-1596, https://doi.org/10.1096/fj.06-6589com. The above article, published online on 31 January 2007, in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Loren E. Wold; the Federation of American Societies for Experimental Biology; and Wiley Periodicals LLC. The retraction has been agreed upon following an investigation into concerns raised by a third party which revealed inappropriate image section duplications within the article (Figures1D, 4B, 5A and B, 6B and C, 7D and 8) and between this (Figure4B) and another article published previously in a different scientific context. Due to the number and the level of errors identified in the published figures, the editors have lost confidence in the presented data and consider the conclusions substantially compromised. The authors have been informed about the concerns, but did not provide an explanation and due to the time elapsed since publication the original raw data are not available.

Releasing a sugar brake generates sweeter tomato without yield penalty

In tomato, sugar content is highly correlated with consumer preferences, with most consumers preferring sweeter fruit1–4. However, the sugar content of commercial varieties is generally low, as it is inversely correlated with fruit size, and growers prioritize yield over flavour quality5–7. Here we identified two genes, tomato (Solanum lycopersicum) calcium-dependent protein kinase 27 (SlCDPK27; also known as SlCPK27) and its paralogue SlCDPK26, that control fruit sugar content. They act as sugar brakes by phosphorylating a sucrose synthase, which promotes degradation of the sucrose synthase. Gene-edited SlCDPK27 and SlCDPK26 knockouts increased glucose and fructose contents by up to 30%, enhancing perceived sweetness without fruit weight or yield penalty. Although there are fewer, lighter seeds in the mutants, they exhibit normal germination. Together, these findings provide insight into the regulatory mechanisms controlling fruit sugar accumulation in tomato and offer opportunities to increase sugar content in large-fruited cultivars without sacrificing size and yield.

CSIG-22. PHARMACOLOGIC INHIBITION AND KNOCKDOWN OF CAMK2 IMPAIR GROWTH OF PATIENT-DERIVED GLIOBLASTOMA CULTURES

Abstract INTRODUCTION Glioblastoma (GBM), a primary brain malignancy with a median survival of 15-18 months, presents a pressing challenge in neuro-oncology. Patient-derived GBM cultures (PDGCs) exhibit spontaneous calcium (Ca2+) waves, which are thought to drive tumor growth and represent a therapeutic target. The mechanisms underlying the generation and the effectors of these Ca2+ waves remain elusive. We hypothesize CAMK2, a Ca2+/calmodulin-dependent protein kinase expressed as four distinct isoforms, is a key effector of such Ca2+ waves by phosphorylating substrates that promote tumor growth. METHODS Using multiple PDGCs, CAMK2 isoform-specific mRNA levels were assessed via qRT-PCR both at baseline and after shRNA-mediated knockdown (KD). Tumorsphere formation and WST-8 assays were performed to assess PDGC clonogenic potential after either KD or pharmacologic inhibition with the cell-permeable CAMK2 inhibitor KN93. RESULTS The relative mRNA levels of CAMK2 isoforms across three PDGC lines were, in decreasing order: CAMK2D, CAMK2G, CAMK2B, and CAMK2A. Expression of CAMK2D- and CAMK2B-specific shRNAs significantly reduced tumorsphere growth compared to control shRNA. CAMK2D KD reduced sphere formation by 55.13 ± 3.54% (p=0.0001) and 47.72 ± 9.55% (p=0.008) in separate PDGC lines, while CAMK2B KD reduced sphere formation by 41.15 ± 11.51% (p=0.021) and sphere size by 41.78% ± 10.50% (p=0.049) (n=3/experiment). KN93 treatment reduced tumorsphere formation by 59.23 ± 1.40% (p=0.0006) and 42.94 ± 13.0% (p=0.032) in two PDGC lines (n=3) and PDGC viability, while CAMK2-inactive analogue KN92 and DMSO minimally impacted viability (p<0.0001, two-way ANOVA). CONCLUSION CAMK2D and CAMK2B KD, as well as KN93 treatment, reduce tumorsphere formation, suggesting CAMK2 has a tumorigenic role in GBM. Further investigation of CAMK2 isoforms and in vivo validation are underway. Future aims include directly testing the link between spontaneous Ca2+ waves and CAMK2 activation and identifying CAMK2 phosphorylation targets that mediate tumor growth.

Establishment of potential lncRNA-related hub genes involved competitive endogenous RNA in lung adenocarcinoma

Long non-coding RNAs (lncRNAs) have a notable role in the diagnosis and prognosis of cancer. However, the associations between lncRNA-related hub genes (LRHGs) expression and the corresponding outcomes have not been fully understood in lung adenocarcinoma (LUAD). Here, a total of 71 patients diagnosed with LUAD and 60 healthy volunteers at The First Affiliated Hospital of Huzhou University from April, 2023 to December, 2023 were enrolled in the present study. A LRHGs model was established using least absolute shrinkage and selection operator analyses of The Cancer Genome Atlas-LUAD datasets. The underlying mechanisms of the LRHGs were investigated via Gene Set Enrichment Analysis and Gene Set Variation Analysis. Additionally, the diagnostic role of serum HOXD cluster antisense RNA 2 (HOXD-AS2) was assessed by receiver operating characteristic (ROC) curve analysis. Lastly, TCGA-LUAD samples were divided into high- and low-HOXD-AS2 expression groups based on the median expression. The associations between HOXD-AS2 expression and miR-4538 as well as Calmodulin-Dependent Protein Kinase Type II subunit Beta (CAMK2B) levels were conducted through Pearson correlation analysis. A comprehensive analysis identified 141 differentially expressed lncRNAs between 539 LUAD tissues and 59 normal samples. A prognostic marker for overall survival was established by constructing a predictive signature consisting of 9 LRHGs. Subsequently, 474 LUAD samples were categorized into a high or low-risk group based on the median of the risk score. An independent prognostic model was constructed to confirm the validity of this categorization. Further comparisons of the clinicopathological features and LRHG-related pathways were performed between the two groups. Examinations of LRHG expression in two LUAD clusters and of the association between LRHG expression and immune infiltration were also conducted. HOXD-AS2 expression was shown to be elevated in LUAD tissues compared with matched normal tissues, and the serum HOXD-AS2 level was also notably increased in LUAD samples compared with healthy controls. The results of the ROC analysis indicated that the sensitivity and specificity of HOXD-AS2 were higher than that of cytokeratin-19 fragment (CYFRA21-1), which is a serum marker for LUAD. Pearson analyses indicated that miR-4538 level was negatively associated with HOXD-AS2 expression, but CAMK2B level showed positive correlation in LUAD. The results of the present study therefore indicated that the constructed LRHG model, particularly HOXD-AS2, could independently diagnose and predict the prognosis of LUAD, which suggested the underlying mechanism of the HOXD-AS2/miR-4538/CAMK2B, and might offer efficient strategies for LUAD treatment.

NMDA receptor blockade attenuates Japanese encephalitis virus infection-induced microglia activation

Neurodegeneration and neuroinflammation are key components in the pathogenesis of Japanese Encephalitis caused by Japanese Encephalitis Virus (JEV) infection. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotoxic and pro-inflammatory properties in a cell context-dependent manner. Herein, potential roles of the NMDA receptor in excitatory neurotoxicity and neuroinflammation and effects of NMDA receptor blockade against JEV pathogenesis were investigated in rat microglia, neuron/glia, neuron cultures, and C57BL/6 mice. In microglia, JEV infection induced glutamate release and activated post-receptor NMDA signaling, leading to activation of Ca2+ mobilization and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), accompanied by pro-inflammatory NF-κB and AP-1 activation and cytokine expression. Additionally, increased Dynamin-Related Protein-1 protein phosphorylation, NAPDH Oxidase-2/4 expression, free radical generation, and Endoplasmic Reticulum stress paralleled with the reactive changes of microglia after JEV infection. JEV infection-induced biochemical and molecular changes contributed to microglia reactivity and pro-inflammatory cytokine expression. NMDA receptor antagonists MK801 and memantine alleviated intracellular signaling and pro-inflammatory cytokine expression in JEV-infected microglia. JEV infection induced neuronal cell death in neuron/glia culture associated with the concurrent production of pro-inflammatory cytokines. Conditioned media of JEV-infected microglia compromised neuron viability in neuron culture. JEV infection-associated neuronal cell death was alleviated by MK801 and memantine. Activation of NMDA receptor-related inflammatory changes, microglia activation, and neurodegeneration as well as reversal effects of memantine were revealed in the brains of JEV-infected mice. The current findings highlight a crucial role of the glutamate/NMDA receptor axis in linking excitotoxicity and neuroinflammation during the course of JEV pathogenesis, and proposes the anti-inflammatory and neuroprotective potential of NMDA receptor blockade.

The ZmCPK39-ZmDi19-ZmPR10 immune module regulates quantitative resistance to multiple foliar diseases in maize.

Gray leaf spot, northern leaf blight and southern leaf blight are three of the most destructive foliar diseases affecting maize (Zea mays L.). Here we identified a gene, ZmCPK39, that encodes a calcium-dependent protein kinase and negatively regulates quantitative resistance to these three diseases. The ZmCPK39 allele in the resistant line displayed significantly lower pathogen-induced gene expression than that in the susceptible line. A marked decrease in ZmCPK39 abundance mitigated the phosphorylation and degradation of the transcription factor ZmDi19. This led to elevated expression of ZmPR10, a gene known to encode an antimicrobial protein, thereby enhancing maize resistance to foliar diseases. Moreover, the F1 hybrid with reduced ZmCPK39 expression favored disease resistance, thereby increasing yield. Hence, the discovery of the ZmCPK39-ZmDi19-ZmPR10 immune module provides insight into the mechanisms underlying broad-spectrum quantitative disease resistance and also offers a new avenue for the genetic control of maize foliar diseases.

CPK28-mediated phosphorylation enhances nitrate transport activity of NRT2.1 during nitrogen deprivation.

Nitrate (NO3 -) serves as the primary inorganic nitrogen source assimilated by most terrestrial plants. The acquisition of nitrate from the soil is facilitated by NITRATE TRANSPORTERS (NRTs), with NRT2.1 being the key high-affinity nitrate transporter. The activity of NRT2.1, which has multiple potential phosphorylation sites, is intricately regulated under various physiological conditions. Here, we discovered that CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) positively regulates nitrate uptake under nitrogen deprivation conditions. We found CPK28 as the kinase targeted by immunoprecipitation followed by mass spectrometry and examined the in-planta phosphorylation status of NRT2.1 in cpk28 mutant plants by employing quantitative MS-based phosphoproteomics. Through a combination of in vitro phosphorylation experiment and immunoblotting using phospho-specific antibody, we successfully demonstrated that CPK28 specifically phosphorylates NRT2.1 at Ser21. Functional analysis conducted in Xenopus oocytes revealed that co-expression of CPK28 significantly enhanced high-affinity nitrate uptake of NRT2.1. Further investigation using transgenic plants showed that the phosphomimic variant NRT2.1S21E, but not the nonphosphorylatable variant NRT2.1S21A, fully restored high-affinity 15NO3 - uptake ability in both nrt2.1 and cpk28 mutant backgrounds. This study clarifies that the kinase activity of CPK28 is promoted during nitrogen deprivation conditions. These significant findings provide valuable insights into the intricate regulatory mechanisms that govern nitrate-demand adaptation.

CaMKII Exacerbates Doxorubicin-Induced Cardiotoxicity by Promoting Ubiquitination Through USP10 Inhibition.

Doxorubicin (DOX) is an effective anticancer drug, but it has a problem of cardiotoxicity that cannot be ignored. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is tightly associated with the pathological progression of DOX-induced cardiotoxicity. Ubiquitin-specific protease 10 (USP10) plays an important role in many biological processes and cancers. However, its association with DOX-induced cardiotoxicity and CaMKII remains unclear. H9C2 cells, HL-1 cells and C57BL/6 mice were used to establish the DOX-induced cardiotoxicity model, and the CaMKII-specific inhibitor KN-93 and USP10 specific inhibitor Spautin-1 were used to observe the CaMKII and USP10 effect. In cell experiments, CCK-8 method was used to assess cell viability, LDH kit was used to assess lactate dehydrogenase expression, DCFH-DA staining was used to observe changes in active oxygen content, TUNEL staining was used to observe cell apoptosis, and Western blotting method was used to detect relevant protein markers. The expression of p-CaMKII and USP10 was assessed by immunofluorescence staining. In animal experiments, mouse echocardiograph was used were used to evaluate cardiac function, and HE staining and Masson staining were used to evaluate myocardial injury. Cardiomyocyte apoptosis was detected by TUNEL staining. Western blotting method was used to detect relevant protein markers. Our results demonstrated that activation of CaMKII and inhibition of USP10 pathway related to DOX-induced cardiotoxicity. Inhibition of CaMKII with KN-93 ameliorated DOX-induced cardiac dysfunction and cytotoxicity. In addition, CaMKII inhibition prevented DOX-induced apoptosis and ubiquitination. Furthermore, CaMKII inhibition increased USP10 expression in DOX-treated mouse hearts, H9C2 cells and HL-1 cells. At last, the USP10 inhibitor, Spautin-1, blocked the regulatory effect of CaMKII inhibition on apoptosis and ubiquitination in DOX-induced cardiotoxicity. Our findings revealed that DOX-induced myocardial apoptosis and activated CaMKII through cellular and animal levels, while providing a novel probe into the mechanism of CaMKII action: promoting ubiquitination by inhibiting USP10 aggravated apoptosis.

Schaftoside improves HFpEF through regulation the autophagy-lysosome pathway by allosterically targeting CaMKII-δ

Heart failure with preserved ejection fraction (HFpEF) presents a significant challenge to global healthcare systems due to its complex presentation. HFpEF presents with a normal or near-normal left ventricular ejection fraction, cardiac diastolic dysfunction, and a metabolic profile characterized by impaired inflammation and oxidative stress. There have been few valuable drug targets reported for HFpEF to date. Here, we discovered that schaftoside, an active component from licorice, has a significant protective effect on the cardiac remodeling induced by continuous infusion of angiotensin II (AngII), which leads to the HFpEF phenotype. Mechanistically, schaftoside has demonstrated the ability to ameliorate lysosomal dysfunction in both in vitro and in vivo models, thereby activating autophagy. Bioinformatic analyses based on proteome and phosphoproteome suggested that Ca2+/calmodulin-dependent protein kinase II (CaMKII) was a potential target for schaftoside. It was confirmed that schaftoside allosterically mediated CaMKII-δ conformation via targeting a unique active pocket near the ATP-binding site to inhibit protein phosphorylation and regulate the lysosomal autophagy pathway. Therefore, schaftoside represents the first small molecule identified to inhibit CaMKII-δ activity through allosteric inhibition, providing a novel candidate for alleviating cardiac metabolic imbalance in HFpEF.