Calmodulin-dependent Kinase Research Articles

Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons.

N-methyl-D-aspartate receptor (NMDAR) trafficking is a key process in the regulation of synaptic efficacy and brain function. However, the molecular mechanism underlying the surface transport of NMDARs is largely unknown. Here we identified myosin Va (MyoVa) as the specific motor protein that traffics NMDARs in hippocampal neurons. We found that MyoVa associates with NMDARs through its cargo binding domain. This association was increased during NMDAR surface transport. Knockdown of MyoVa suppressed NMDAR transport. We further demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates NMDAR transport through its direct interaction with MyoVa. Furthermore, MyoVa employed Rab11 family-interacting protein 3 (Rab11/FIP3) as the adaptor proteins to couple themselves with NMDARs during their transport. Accordingly, the knockdown of FIP3 impairs hippocampal memory. Together, we conclude that in hippocampal neurons, MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner.

Kinase signalling adaptation supports dysfunctional mitochondria in disease.

Mitochondria form a critical control nexus which are essential for maintaining correct tissue homeostasis. An increasing number of studies have identified dysregulation of mitochondria as a driver in cancer. However, which pathways support and promote this adapted mitochondrial function? A key hallmark of cancer is perturbation of kinase signalling pathways. These pathways include mitogen activated protein kinases (MAPK), lipid secondary messenger networks, cyclic-AMP-activated (cAMP)/AMP-activated kinases (AMPK), and Ca2+/calmodulin-dependent protein kinase (CaMK) networks. These signalling pathways have multiple substrates which support initiation and persistence of cancer. Many of these are involved in the regulation of mitochondrial morphology, mitochondrial apoptosis, mitochondrial calcium homeostasis, mitochondrial associated membranes (MAMs), and retrograde ROS signalling. This review will aim to both explore how kinase signalling integrates with these critical mitochondrial pathways and highlight how these systems can be usurped to support the development of disease. In addition, we will identify areas which require further investigation to fully understand the complexities of these regulatory interactions. Overall, this review will emphasize how studying the interaction between kinase signalling and mitochondria improves our understanding of mitochondrial homeostasis and can yield novel therapeutic targets to treat disease.

Sugar coordinates plant defense signaling.

Pathogen recognition triggers energy-intensive defense systems. Although successful defense should depend on energy availability, how metabolic information is communicated to defense remains unclear. We show that sugar, especially glucose-6-phosphate (G6P), is critical in coordinating defense in Arabidopsis. Under sugar-sufficient conditions, phosphorylation levels of calcium-dependent protein kinase 5 (CPK5) are elevated by G6P-mediated suppression of protein phosphatases, enhancing defense responses before pathogen invasion. Subsequently, recognition of bacterial flagellin activates sugar transporters, leading to increased cellular G6P, which elicits CPK5-independent signaling promoting synthesis of the phytohormone salicylic acid (SA) for antibacterial defense. In contrast, while perception of fungal chitin does not promote sugar influx or SA accumulation, chitin-induced synthesis of the antifungal compound camalexin requires basal sugar influx activity. By monitoring sugar levels, plants determine defense levels and execute appropriate outputs against bacterial and fungal pathogens. Together, our findings provide a comprehensive view of the roles of sugar in defense.

Biological evaluation of novel side chain containing CQTrICh-analogs as antimalarials and their development as PfCDPK1 kinase inhibitors

The rapid emergence of resistance to existing frontline antimalarial drugs emphasizes a need for the development of target-oriented molecules with novel modes of action. Given the importance of a plant-like Calcium-Dependent Protein Kinase 1 (PfCDPK1) as a stand-alone multistage signalling regulator of P. falciparum, we designed and synthesized 7-chloroquinoline-indole-chalcones tethered with a triazole (CQTrICh-analogs 7 (a–s) and 9) directed towards PfCDPK1. This was accomplished by reacting substituted 1-phenyl-3-(1-(prop-2-yn-1-yl)-1H-indol-3-yl) prop-2-en-1-one and 1-(prop-2-yn-1-yl)-1H-indole-3-carbaldehyde with 4-azido-7-chloroquinoline, respectively via a ‘click’ reaction. The selected CQTrICh-analogs: 7l and 7r inhibited the growth of chloroquine-sensitive 3D7 strain and -resistant RKL-9 isolate of Plasmodium falciparum, with IC50 values of 2.4 μM &1.8 μM (7l), and 3.5 μM &2.7 μM (7r), respectively, and showed no apparent hemolytic activity and cytotoxicity in mammalian cells. Intra-erythrocytic progression studies revealed that the active hybrids: 7l and 7r are effective against the mature stages of the parasite. 7l and 7r were found to stably interact with the catalytically active ATP-binding pocket of PfCDPK1 via energetically favourable H-bonds. The interaction was confirmed in vitro by microscale thermophoresis and kinase assays, which demonstrated that the active hybrids interact with PfCDPK1 and inhibit its kinase activity which is presumably responsible for the parasite growth inhibition. Interestingly, 7l and 7r showed no inhibitory effect on the human kinases, indicating their selectivity for the parasite kinase. We report the antiplasmodial potential of novel kinase-targeting bio-conjugates, a step towards developing pan-kinase inhibitors which is a prerequisite for multistage anti-malarial protection.

Ilex paraguariensis A.St.-Hil. improves lipid metabolism in high-fat diet-fed obese rats and suppresses intracellular lipid accumulation in 3T3-L1 adipocytes via the AMPK-dependent and insulin signaling pathways.

Obesity is closely associated with several chronic diseases, and adipose tissue plays a major role in modulating energy metabolism. This study aimed to determine whether Mate, derived from I. paraguariensis A.St.-Hil., ameliorates lipid metabolism in 3T3-L1 adipocytes and high-fat diet (HFD)-fed obese Sprague-Dawley (SD) rats. 3T3-L1 adipocytes were cultured for 7 days, following which intracellular lipid accumulation and expression levels of lipid metabolism-related factors were examined. Dorsomorphin was used to investigate the potential pathways involved, particularly the adenosine monophosphate-activated protein kinase (AMPK)- dependent pathway. Mate was administered to rat HFD-fed obese SD models for 8 consecutive weeks. The expression of lipid metabolism-related factors in the organs and tissues collected from dissected SD rats was evaluated. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes, increased the protein and gene expression levels of AMPK, hormone sensitive lipase (HSL), calmodulin kinase kinase (CaMKK), liver kinase B1 (LKB1), protein kinase A (PKA), CCAAT/enhancer binding protein β (C/EBPβ), insulin receptor b (IRβ), and insulin receptor substrate 1 (IRS1) (Tyr465), and decreased those of sterol regulatory element binding protein 1C (Srebp1c), fatty acid synthase (FAS), peroxisome-activated receptor γ (PPARγ), and IRS1 (Ser1101). Furthermore, an AMPK inhibitor abolished the effects exerted by Mate on intracellular lipid accumulation and HSL and FAS expression levels. Mate treatment suppressed body weight gain and improved serum cholesterol levels in HFD-fed obese SD rats. Treatment with Mate increased the protein and gene expression levels of AMPK, PKA, Erk1/Erk2 (p44/p42), and uncoupling protein 1 and reduced those of mammalian target of rapamycin, S6 kinase, Srebp1c, ap2, FAS, Il6, Adiponectin, Leptin, and Fabp4 in rat HFD-fed obese SD models. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes and improved lipid metabolism in the epididymal adipose tissue of HFD-fed obese SD rats via the activation of AMPK-dependent and insulin signaling pathways.

The LKB1–TSSK1B axis controls YAP phosphorylation to regulate the Hippo–YAP pathway

The Hippo pathway’s main effector, Yes-associated protein (YAP), plays a crucial role in tumorigenesis as a transcriptional coactivator. YAP’s phosphorylation by core upstream components of the Hippo pathway, such as mammalian Ste20 kinase 1/2 (MST1/2), mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), and their substrate, large tumor suppressor 1/2 (LATS1/2), influences YAP’s subcellular localization, stability, and transcriptional activity. However, recent research suggests the existence of alternative pathways that phosphorylate YAP, independent of these core upstream Hippo pathway components, raising questions about additional means to inactivate YAP. In this study, we present evidence demonstrating that TSSK1B, a calcium/calmodulin-dependent protein kinase (CAMK) superfamily member, is a negative regulator of YAP, suppressing cellular proliferation and oncogenic transformation. Mechanistically, TSSK1B inhibits YAP through two distinct pathways. Firstly, the LKB1–TSSK1B axis directly phosphorylates YAP at Ser94, inhibiting the YAP–TEAD complex’s formation and suppressing its target genes’ expression. Secondly, the TSSK1B–LATS1/2 axis inhibits YAP via phosphorylation at Ser127. Our findings reveal the involvement of TSSK1B-mediated molecular mechanisms in the Hippo–YAP pathway, emphasizing the importance of multilevel regulation in critical cellular decision-making processes.

Interleukin-33/ST2 axis involvement in atrial remodeling and arrhythmogenesis

Interleukin (IL)-33, a cytokine involved in immune responses, can activate its receptor, suppression of tumorigenicity 2 (ST2), is elevated during atrial fibrillation (AF). However, the role of IL-33/ST2 signaling in atrial arrhythmia is unclear. This study explored the pathological effects of the IL-33/ST2 axis on atrial remodeling and arrhythmogenesis. Patch clamping, confocal microscopy, and Western blotting were used to analyze the electrical characteristics of and protein activity in atrial myocytes (HL-1) treated with recombinant IL-33 protein and/or ST2-neutralizing antibodies for 48 hrs. Telemetric electrocardiographic recordings, Masson's trichrome staining, and immunohistochemistry staining of the atrium were performed in mice receiving tail vein injections with nonspecific immunoglobulin (control), IL-33, and IL-33 combined with anti-ST2 antibody for 2 weeks. IL-33-treated HL-1 cells had a reduced action potential duration, lower L-type Ca2+ current, greater sarcoplasmic reticulum (SR) Ca2+ content, increased Na+/Ca2+ exchanger (NCX) current, elevation of K+ currents, and increased intracellular calcium transient. IL-33-treated HL-1 myocytes had greater activation of the calcium–calmodulin-dependent protein kinase II (CaMKII)/ryanodine receptor 2 (RyR2) axis and nuclear factor kappa B (NF-κB) / NLR family pyrin domain containing 3 (NLRP3) signaling than did control cells. IL-33 treated cells also had greater expression of Nav1.5, Kv1.5, NCX, and NLRP3 than did control cells. Pretreatment with neutralizing anti-ST2 antibody attenuated IL-33-mediated activation of CaMKII/RyR2 and NF-κB/NLRP3 signaling. IL-33-injected mice had more atrial ectopic beats and increased AF episodes, greater atrial fibrosis, and elevation of NF-κB/NLRP3 signaling than did controls or mice treated with IL-33 combined with anti-ST2 antibody. Thus, IL-33 recombinant protein treatment promotes atrial remodeling through ST2 signaling. Blocking the IL-33/ST2 axis might be an innovative therapeutic approach for patients with atrial arrhythmia and elevated serum IL-33.

8-Br-cGMP suppresses tumor progression through EGFR/PLC γ1 pathway in epithelial ovarian cancer.

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (PKG-I), a serine/threonine kinase, is important in tumor development. The present study determines that the cGMP/PKG I pathway is essential for promoting cell proliferation and survival in human ovarian cancer cells, whereas cGMP analog has been shown to lead to growth inhibition and apoptosis of various cancer cells. The role of cGMP/PKG I pathway in epithelial ovarian cancer (EOC), therefore, remains controversial. We investigated the effect of cGMP/PKG I pathway and the underlying mechanism in EOC. The results showed that exogenous 8-Bromoguanosine-3', 5'-cyclic monophosphate (8-Br-cGMP) (cGMP analog) could antagonize the effects by EGF, including suppressing proliferation, invasion and migration of EOC cells. In vivo, 8-Br-cGMP hampered the growth of the xenograft tumor. Additionally, the expressions of epidermal growth factor receptor (EGFR), matrix metallopeptidase 9 (MMP9), proliferating cell nuclear antigen and Ki67 in xenograft tumor were decreased after 8-Br-cGMP intervention. Further research demonstrated that 8-Br-cGMP decreased the phosphorylation of EGFR (Y992) and downstream proteins phospholipase Cγ1 (PLC γ1) (Y783), calmodulin kinase II (T286) and inhibited cytoplasmic Ca2+ release as well as PKC transferring to cell membrane. It's worth noting that the inhibition was 8-Br-cGMP dose-dependent and 8-Br-cGMP showed similar inhibitory effect on EOC cells compared with U-73122, a specific inhibitor of PLC γ1. The activation of endogenous PKG I by addition of exogenous 8-Br-cGMP could inhibit EOC development probably via EGFR/PLCγ1 signaling pathway. 8-Br-cGMP/PKG I provide a new insight and strategy for EOC treatment.

CaMKIIδ, Stabilized by RNA N6-Methyladenosine Reader IGF2BP2, Boosts Coxsackievirus B3-Induced Myocardial Inflammation via Interacting with TIRAP.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.

Cigarette tar accelerates atherosclerosis progression via RIPK3-dependent necroptosis mediated by endoplasmic reticulum stress in vascular smooth muscle cells

BackgroundTar is the main toxic of cigarettes, and its effect on atherosclerosis progression and the underlying mechanisms remain largely unknown. Vascular smooth muscle cells (VSMCs) play a key role in atherogenesis and plaque vulnerability. The present study sought to investigate the mechanism of atherosclerosis progression through tar-induced VSMC necroptosis, a recently described form of necrosis.MethodsThe effect of tar on atherosclerosis progression and VSMC necroptosis was examined in ApoE−/− mice and cultured VSMCs. The role of necroptosis in tar-induced plaque development was evaluated in RIPK3-deletion mice (ApoE−/−RIPK3−/−). The key proteins of necroptosis in carotid plaques of smokers and non-smokers were also examined. Quantitative proteomics of mice aortas was conducted to further investigate the underlying mechanism. Pharmacological approaches were then applied to modulate the expression of targets to verify the regulatory process of tar-induced necroptosis.ResultsTar administration led to increased atherosclerotic plaque area and reduced collagen and VSMCs in ApoE−/− mice. The expression of RIPK1、RIPK3、and MLKL in VSMCs of plaques were all increased in tar-exposed mice and smokers. RIPK3 deletion protected against VSMC loss and plaque progression stimulated by tar. In mechanistic studies, quantitative proteomics analysis of ApoE−/− mice aortas suggested that tar triggered endoplasmic reticulum (ER) stress. PERK-eIF2α-CHOP axis was activated in tar-treated VSMCs and atherosclerotic plaque. Inhibition of ER stress using 4PBA significantly reduced plaque progression and VSMC necroptosis. Further study revealed that ER stress resulted in calcium (Ca2+) release into mitochondria and cytoplasm. Elevated Ca2+ levels lead to mitochondrial dysfunction and excessive reactive oxygen species (ROS) production, which consequently promote RIPK3-dependent necroptosis. In addition, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activated by cytosolic Ca2+ overload binds to RIPK3, accounting for necroptosis.ConclusionThe findings revealed that cigarette tar promoted atherosclerosis progression by inducing RIPK3-dependent VSMC necroptosis and identified novel avenues of ER stress and Ca2+ overload.

A Novel Druggable Dual-Specificity tYrosine-Regulated Kinase3/Calmodulin Kinase-like Vesicle-Associated Signaling Module with Therapeutic Implications in Neuroblastoma.

High-risk neuroblastoma is a very aggressive pediatric cancer, accounting for ~15% of childhood cancer mortality. Therefore, novel therapeutic strategies for the treatment of neuroblastoma are urgently sought. Here, we focused on the potential implications of the Dual-specificity tYrosine-Regulated Kinase (DYRK) family and downstream signaling pathways. We used bioinformatic analysis of public datasets from neuroblastoma cohorts and cell lines to search correlations between patient survival and expression of DYRK kinases. Additionally, we performed biochemical, molecular, and cellular approaches to validate and characterize our observations, as well as an in vivo orthotopic murine model of neuroblastoma. We identified the DYRK3 kinase as a critical mediator of neuroblastoma cell proliferation and in vivo tumor growth. DYRK3 has recently emerged as a key regulator of several biomolecular condensates and has been linked to the hypoxic response of neuroblastoma cells. Our data suggest a role for DYRK3 as a regulator of the neuroblastoma-specific protein CAMKV, which is also required for neuroblastoma cell proliferation. CAMKV is a very understudied member of the Ca2+/calmodulin-dependent protein kinase family, originally described as a pseudokinase. We show that CAMKV is phosphorylated by DYRK3, and that inhibition of DYRK3 kinase activity induces CAMKV aggregation, probably mediated by its highly disordered C-terminal half. Importantly, we provide evidence that the DYRK3/CAMKV signaling module could play an important role for the function of the mitotic spindle during cell division. Our data strongly support the idea that inhibition of DYRK3 and/or CAMKV in neuroblastoma cells could constitute an innovative and highly specific intervention to fight against this dreadful cancer.

The Calcium-Dependent Protein Kinase TaCDPK7 Positively Regulates Wheat Resistance to Puccinia striiformis f. sp. tritici.

Ca2+ plays a crucial role as a secondary messenger in plant development and response to abiotic/biotic stressors. Calcium-dependent protein kinases (CDPKs/CPKs) are essential Ca2+ sensors that can convert Ca2+ signals into downstream phosphorylation signals. However, there is limited research on the function of CDPKs in the context of wheat-Puccinia striiformis f. sp. tritici (Pst) interaction. In this study, we aimed to address this gap by identifying putative CDPK genes from the wheat reference genome and organizing them into four phylogenetic clusters (I-IV). To investigate the expression patterns of the TaCDPK family during the wheat-Pst interaction, we analyzed time series RNA-seq data and further validated the results through qRT-PCR assays. Among the TaCDPK genes, TaCDPK7 exhibited a significant induction during the wheat-Pst interaction, suggesting that it has a potential role in wheat resistance to Pst. To gain further insights into the function of TaCDPK7, we employed virus-induced gene silencing (VIGS) to knock down its expression which resulted in impaired wheat resistance to Pst, accompanied by decreased accumulation of hydrogen peroxide (H2O2), increased fungal biomass ratio, reduced expression of defense-related genes, and enhanced pathogen hyphal growth. These findings collectively suggest that TaCDPK7 plays an important role in wheat resistance to Pst. In summary, this study expands our understanding of wheat CDPKs and provides novel insights into their involvement in the wheat-Pst interaction.

Proteomic and molecular analyses to understand the promotive effect of safranal on soybean growth under salt stress

Safranal is a free radical scavenger and useful as an antioxidant molecule; however, its promotive role in soybean is not explored. Salt stress decreased soybean growth and safranal improved it even if under salt stress. To study the positive mechanism of safranal on soybean growth, a proteomic approach was used. According to functional categorization, oppositely changed proteins were further confirmed using biochemical techniques. Actin and calcium-dependent protein kinase decreased in soybean root and hypocotyl, respectively, under salt stress and increased with safranal application. Xyloglucan endotransglucosylase/ hydrolase increased in soybean root under salt stress but decreased with safranal application. Peroxidase increased under salt stress and further enhanced by safranal application in soybean root. Actin, RuvB-like helicase, and protein kinase domain-containing protein were upregulated under salt stress and further enhanced by safranal application under salt stress. Dynamin GTPase was downregulated under salt stress but recovered with safranal application under salt stress. Glutathione peroxidase and PfkB domain-containing protein were upregulated by safranal application under salt stress in soybean root. These results suggest that safranal improves soybean growth through the regulation of cell wall and nuclear proteins along with reactive‑oxygen species scavenging system. Furthermore, it might promote salt-stress tolerance through the regulation of membrane proteins involved in endocytosis and post-Golgi trafficking. SignificanceTo study the positive mechanism of safranal on soybean growth, a proteomic approach was used. According to functional categorization, oppositely changed proteins were further confirmed using biochemical techniques. Actin and calcium-dependent protein kinase decreased in soybean root and hypocotyl, respectively, under salt stress and increased with safranal application. Xyloglucan endotransglucosylase/ hydrolase increased in soybean root under salt stress but decreased with safranal application. Peroxidase increased under salt stress and further enhanced by safranal application in soybean root. Actin, RuvB-like helicase, and protein kinase domain-containing protein were upregulated under salt stress and further enhanced by safranal application under salt stress. Dynamin GTPase was downregulated under salt stress but recovered with safranal application under salt stress. Glutathione peroxidase and PfkB domain-containing protein were upregulated by safranal application under salt stress in soybean root. These results suggest that safranal improves soybean growth through the regulation of cell wall and nuclear proteins along with reactive‑oxygen species scavenging system. Furthermore, it might promote salt-stress tolerance through the regulation of membrane proteins involved in endocytosis and post-Golgi trafficking.

Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress

BackgroundEarly life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. MethodsMale Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. ResultsWe report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LimitationsThe study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. ConclusionDouble-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.

Secretoneurin levels are higher in dilated cardiomyopathy than in ischaemic cardiomyopathy: preliminary results.

Secretoneurin (SN) is a neuropeptide with potential utility as a biomarker of cardiovascular episodes. The main effect of SN is mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences calcium handling. We aimed to associate the levels of SN in plasma with different causes of heart failure. We prospectively enrolled consecutive patients with ischaemic (ICM) and dilated (DCM) cardiomyopathy from the outpatient heart failure clinic and healthy individuals. SN was analysed from venous blood by use of the ELISA method. SN plasma levels were compared in DCM, ICM and healthy individuals with non-parametric tests. A total of 53 patients (81.1% male, 18.9% female; mean age 67.9 ± 12.6 years) and 34 healthy individuals (38% male, 62% female) were included in the analysis. Plasma SN levels were significantly higher in the dilated cardiomyopathy (38.8 ± 27 pmol/L) as compared with the ischaemic cardiomyopathy (19.7 ± 22.6 pmol/L) group (P = 0.006). There was no significant difference between females vs. males (27.1 ± 23 vs. 25.5 ± 26.2 pmol/L, P = NS). Plasma SN levels allowed DCM and ICM to be differentiated with 88% sensitivity and 61% specificity (P = 0.007), the cut of value is 13.3 pmol/L. Plasma SN levels differed significantly between healthy volunteers and both ICM (P < 0.0001) and DCM (P = 0.049). Plasma SN levels did not differ according to age and were not associated with comorbidities, left ventricular ejection fraction, heart failure medication, troponin, creatinine, or natriuretic peptide plasma levels. Plasma secretoneurin levels differed significantly in DCM vs. ICM, being higher in the former. Based on plasma SN levels, discrimination between DCM and ICM might be possible. Healthy individuals produce higher SN plasma levels than stable HFrEF patients.

Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model.

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus.

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

Repetitive Transcranial Magnetic Stimulation Alleviates MPTP-Induced Parkinson's Disease Symptoms by Regulating CaMKII-CREB-BMAL1 Pathway in Mice Model.

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive neuromodulation technique that shows promise for the treatment of Parkinson's disease (PD). However, there is still limited understanding of the optimal stimulation frequencies and whether rTMS can alleviate PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. A PD mouse model was induced intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and treated with 1 Hz, 5 Hz, and 10 Hz rTMS. The neurological function, survival of dopaminergic neurons, and protein levels of Tyrosine hydroxylase (TH), α-synuclein(α-syn), and brain-derived neurotrophic factor (BDNF) in the striatum were measured to determine the optimal stimulation frequencies of rTMS treatment in PD mice. The levels of melatonin, cortisol, and the circadian rhythm of Brain and muscle ARNT-like 1 (BMAL1) in PD model mice were detected after optimal frequency rTMS treatment. Additionally, KN-93 and Bmal1siRNA interventions were used to verify that rTMS could alleviate PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. Administration of 10 Hz rTMS significantly improved neurological function, increased the protein levels of TH and BDNF, and inhibited abnormal aggregation of a-syn. Furthermore, administration of 10 Hz rTMS regulated the secretion profile of cortisol and melatonin and reversed the circadian arrhythmia of BMAL1 expression. After the KN-93 intervention, the MPTP+rTMS+KN-93 group exhibited decreased levels of P- Ca2+/calmodulin-dependent protein kinase II (CaMKII)/CaMKII, P-cAMP-response-element-binding protein (CREB)/CREB, BMALI, and TH. After Bmal1siRNA intervention, the protein levels of BMAL1 and TH were significantly reduced in the MPTP+10 Hz+ Bmal1siRNA group. At the same time, there were no significant changes in the proportions of P-CaMKIIα/CaMKIIα and P-CREB/CREB expression levels. Finally, immunohistochemical analysis showed that the number of TH-positive neurons was high in the MPTP+10 Hz group, but decreased significantly after KN-93 and Bmal1siRNA interventions. Treatment with 10 Hz rTMS alleviated MPTP-induced PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. This study provides a comprehensive perspective of the therapeutic mechanisms of rTMS in PD.

Identification and expression analysis of calcium-dependent protein kinase family in oat (Avena sativa L.) and their functions in response to saline-alkali stresses.

Calcium-dependent protein kinases (CDPKs) serve as calcium ion sensors and play crucial roles in all aspects of plant life cycle. While CDPK gene family has been extensively studied in various plants, there is limited information available for CDPK members in oat, an important cereal crop worldwide. Totally, 60 AsCDPK genes were identified in oat genome and were classified into four subfamilies based on their phylogenetic relationship. The members within each subfamily shared similar gene structure and conserved motifs. Collinearity analysis revealed that AsCDPK gene amplification was attributed to segmental duplication events and underwent strong purifying selection. AsCDPK promoters were predicted to contain cis-acting elements associated with hormones, biotic and abiotic stresses. AsCDPK gene expressions were induced by different salt stresses, exhibiting stress-specific under different salt treatments. Moreover, overexpression of AsCDPK26 gene enhanced salt resistance in C. reinhardtii, a single-cell photoautotrophic model plants. Further analysis revealed a significant correlation between AsCDPK26 and Na+/H+ antiporter 1 (p<0.05), suggesting that AsCDPK26 may interact with ion transporter to modulate salt resistance. These results not only provide valuable insights into AsCDPK genes in response to different salt stresses, but also lay the foundation to mine novel candidates for improving salt tolerance in oat and other crops.