Calmodulin Concentration Research Articles

Anesthetics and Cell-Cell Communication: Potential Ca2+-Calmodulin Role in Gap Junction Channel Gating by Heptanol, Halothane and Isoflurane.

Cell–cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca2+ channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca2+i nor pHi were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca2+ concentration ([Ca2+]i) and consequential activation of calmodulin linked to gap junction proteins.

Kinetic Studies of the Activation of Bordetella pertussis Adenylate Cyclase by Calmodulin.

Adenylate cyclase toxin (ACT) is a virulence factor secreted by Bordetella pertussis and plays a causative role in whooping cough. After ACT attaches to lung phagocytes, the adenylate cyclase (AC) domain of the toxin is transported into the cytoplasm where it is activated by calmodulin (CaM) to cyclize ATP into 3',5'-cyclic adenosine monophosphate (cAMP). Production of high concentrations of cAMP disrupts immune functions of phagocytes. To better understand the mechanism of activation of AC by CaM, the studies reported herein were conducted. Major observations are as follows: (1) dependence of steady-state velocities on CaM and ATP concentrations suggests that CaM and ATP bind to AC in a random fashion. (2) A pre-steady-state lag phase is observed when AC is added to solutions of CaM and ATP, reflecting the association of AC and CaM. Analysis of pre-steady-state data indicates that CaM binds to AC and AC:ATP with second-order rate constants of 30 and 60 μM-1 s-1, respectively, and that CaM dissociates from the resultant complexes with a first-order rate constant of 0.002 s-1. (3) A biphasic dependence of steady-state velocities on CaM concentration is observed: the first phase extending from 0.01 to 1 nM CaM (Kd,obs ∼ 0.06 nM) and the second phase from 1 to 2000 nM CaM (Kd,obs ∼ 60 nM). These results suggest that AC exists in at least two conformations, with each conformation exhibiting distinct binding affinity for CaM and distinct potential for activation.

Study on a Plasmonic Tilted Fiber Grating-Based Biosensor for Calmodulin Detection.

Tilted fiber Bragg grating, which has the advantages of both fiber Bragg grating and long-period fiber grating, has been widely studied for sensing in many fields, especially in the field of biochemistry. Calmodulin, which has a wide distribution in eukaryotes, can regulate several enzymes such as adenylate cyclase and guanylate cyclase and mediates several cellular processes such as cell proliferation and cyclic nucleotide metabolism. The abnormal levels of calmodulin in the body will result in serious effects from metabolism to nerve growth and memory. Therefore, it is important to measure the calmodulin concentration in the body. In this work, we propose and experimentally demonstrate a plasmonic tilted fiber Bragg grating-based biosensor for calmodulin detection. The biosensor was made using an 18° tilted fiber Bragg grating with a 50 nm-thick gold nanofilm coating the surface of the fiber, and transient receptor potential channels were bonded onto the surface of the gold nanofilm to serve as bio-detectors for calmodulin detection. Experimental results showed that the limit of detection using our biosensor was 0.44 nM. Furthermore, we also demonstrated that the interaction between calmodulin and transient receptor potential channels was quite weak without calcium in the solution, which agrees with the biology. Our proposed biosensor has a simple structure, is easy to manufacture, and is of small size, making it a good choice for real-time, label-free, and microliter-volume biomolecule detection.

Electrical therapies act on the Ca2+/CaM signaling pathway to enhance bone regeneration with bioactive glass [S53P4] and allogeneic grafts

This study aimed to investigate the application of low-intensity electrostimulation (ES) and electromagnetic stimulation (EM) associated with bioactive glass (BG) or allogeneic grafts (BB) in bone regeneration. A cell viability test on osteoblasts (UMR-106) was performed in the presence of BB and BG grafts associated with ES (10μA/5min) and EM (500 Hz/2min). Critical defects (25 mm2 ) in calvaria were generated in male Wistar rats, and bone regeneration was evaluated on the 30th, 60th, and 120th days after surgery. Cell proliferation increased with the application of ES in both grafts and after EM with BG. Bone remodeling was more effective using the allogeneic graft in both therapies, with increased angiogenesis, osteoblast proliferation, and OPN expression in the BB + EM group. A higher number of osteoblasts and osteoclasts, and an increase in bone sialoprotein, Runx-2, and Opn gene expression were found in the BB + ES group. The BG graft associated with EM therapy had an increased proliferation of osteoblasts and increased expression of Runx-2 and Opn. Groups that had BG and ES therapy had increased numbers of osteoblasts, osteoclasts, and increased OPN expression. The expression of voltage-gated calcium channels increased in groups with ES, while calmodulin expression increased in therapies without grafting. ES and EM therapies favored the repair of bone defects upon grafting by improving angiogenesis, osteogenic gene expression, and tissue reorganization. Despite activating different pathways, both therapies increased the intracellular concentrations of calmodulin, leading to cell proliferation and bone regeneration.

The Relevance of Amyloid β-Calmodulin Complexation in Neurons and Brain Degeneration in Alzheimer’s Disease

Intraneuronal amyloid β (Aβ) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer’s disease (AD)-affected brains, intraneuronal Aβ oligomers can derive from Aβ peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aβ neurotoxicity in AD. However, the identification of primary Aβ-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aβ peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aβ concentrations in the low nanomolar range. In turn, the concentration of Aβ-CaM complexes within neurons will increase as a function of time after the induction of Aβ production, and free Aβ will rise sharply when accumulated Aβ exceeds all available CaM. Thus, Aβ-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aβ; a point that has been overlooked until now. In this review, we address the implications of Aβ-CaM complexation in the formation of neurotoxic Aβ oligomers, in the alteration of intracellular calcium homeostasis induced by Aβ, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aβ.

Mechanistic link between CaM-RyR2 interactions and the genesis of cardiac arrhythmia

In this study, we develop a computational model of the interaction between ryanodine receptor type 2 (RyR2) and calmodulin (CaM) to explore the mechanistic link between CaM-RyR2 interactions and cardiac arrhythmia. Our starting point is a biophysically based computational model of CaM binding to a single RyR2 subunit, which reproduces single-channel RyR2 measurements in lipid bilayers. We then integrate this CaM-RyR2 model into a spatially distributed whole-cell model of Ca cycling, which is used to investigate the relationship between CaM and Ca cycling homeostasis. We show that a reduction in CaM concentration leads to a substantial increase in the rate of spontaneous Ca sparks, and this induces a marked reduction in sarcoplasmic reticulum Ca load during steady-state pacing. Also, we show that a reduction in CaM modifies the RyR2 open probability, which makes the cell more prone to Ca wave propagation. These results indicate that aberrant Ca cycling activity during pacing is determined by the interplay between sarcoplasmic reticulum load reduction and the threshold for Ca wave propagation. Based on these results, we show that when CaM is reduced, Ca waves can occur in a cell and induce action potential perturbations that are arrhythmogenic. Thus, this study outlines a novel, to our knowledge, mechanistic link between CaM-RyR2 binding kinetics and the induction of arrhythmias in the heart.

Effects of biogenic amines on the immune response and immunoregulation mechanism in hemocytes of Litopenaeus vannamei in vitro

The effects of three biogenic amines (DA, NE and 5-HT) on the immune signaling pathway and immune response of hemocytes in shrimp were investigated through in vitro experiments. The results showed that the G protein effectors (AC, PLC), the second messengers (cAMP, DAG), Calmodulin (CaM) and protein kinases (PKA, PKC) of DA and NE groups shared a similar trend in which all intracellular signaling factors increased significantly and reached the maximum at 3 h. The concentrations of AC, cAMP and PKA in 5-HT groups decreased significantly compared with the control group, while the concentrations of PLC, CaM, DAG and PKC in 5-HT groups increased markedly. The immune parameters such as total hemocyte count (THC), cell viability, antibacterial activity and bacteriolytic activity, as well as prophenoloxidase (proPO) activity in three biogenic amines groups decreased significantly, while the phenoloxidase (PO) activity increased significantly. The phagocytic activity in DA and NE groups decreased significantly, while that in 5-HT groups increased markedly and reached the highest level at 1 h. Among these three biogenic amines, DA showed the strongest effect on the immune activity of the hemocytes, whereas 5-HT had the least effect. In addition, we speculated that DA and NE might regulate phagocytosis by activating intracellular AC-cAMP-PKA pathway while 5-HT might inhibit intracellular AC-cAMP-PKA pathway. Moreover, the activation of proPO system might be related to PLC-DAG-PKC and PLC-CaM pathway.

Interactions between calmodulin and neurogranin govern the dynamics of CaMKII as a leaky integrator.

Calmodulin-dependent kinase II (CaMKII) has long been known to play an important role in learning and memory as well as long term potentiation (LTP). More recently it has been suggested that it might be involved in the time averaging of synaptic signals, which can then lead to the high precision of information stored at a single synapse. However, the role of the scaffolding molecule, neurogranin (Ng), in governing the dynamics of CaMKII is not yet fully understood. In this work, we adopt a rule-based modeling approach through the Monte Carlo method to study the effect of Ca2+ signals on the dynamics of CaMKII phosphorylation in the postsynaptic density (PSD). Calcium surges are observed in synaptic spines during an EPSP and back-propagating action potential due to the opening of NMDA receptors and voltage dependent calcium channels. Using agent-based models, we computationally investigate the dynamics of phosphorylation of CaMKII monomers and dodecameric holoenzymes. The scaffolding molecule, Ng, when present in significant concentration, limits the availability of free calmodulin (CaM), the protein which activates CaMKII in the presence of calcium. We show that Ng plays an important modulatory role in CaMKII phosphorylation following a surge of high calcium concentration. We find a non-intuitive dependence of this effect on CaM concentration that results from the different affinities of CaM for CaMKII depending on the number of calcium ions bound to the former. It has been shown previously that in the absence of phosphatase, CaMKII monomers integrate over Ca2+ signals of certain frequencies through autophosphorylation (Pepke et al, Plos Comp. Bio., 2010). We also study the effect of multiple calcium spikes on CaMKII holoenzyme autophosphorylation, and show that in the presence of phosphatase, CaMKII behaves as a leaky integrator of calcium signals, a result that has been recently observed in vivo. Our models predict that the parameters of this leaky integrator are finely tuned through the interactions of Ng, CaM, CaMKII, and PP1, providing a mechanism to precisely control the sensitivity of synapses to calcium signals. Author Summary not valid for PLOS ONE submissions.

Differential Protein‐Protein Interactions Driven by the Arg/Gly389 Single Nucleotide Polymorphisms in the Cardiac Beta‐1 Adrenergic Receptor

Activation of the β1 adrenergic receptor (β1AR) increases electrically induced calcium signals and accelerates sequestration of calcium in the myocardium, promoting contraction and relaxation. In humans, Arg389 and Gly389 are common single nucleotide polymorphisms (SNPs) in β1AR. The Arg389 SNP is associated with higher responses to β agonists in terms of cAMP production and contractile force development; in living subjects, it also is coupled with faster response to b blockers with respect to reductions in heart rate and myocardial contractile force. Nevertheless, it is unknown if calcium signals following β1AR activation affects the receptor’s activities and changes in Ca2+‐dependent regulation of β1AR can explain the difference between these polymorphisms. Calmodulin is the ubiquitous transducer of calcium signals and is a limiting factor in cardiomyocytes. We tested the hypothesis that β1AR directly interacts with calmodulin and that the Arg389 and Gly389 SNPs are associated with disparate β1AR‐calmodulin interactions. Using novel fluorescent biosensors developed for β1AR, we identified a novel calmodulin‐binding domain located at the juxtamembranous region of β1AR that encompasses the Arg/Gly389 SNP. The Arg389 SNP is associated with a biphasic interaction with Ca2+/calmodulin. However, the Gly389 SNP is associated with a monophasic interaction with CaM and a ~12‐fold lower affinity than the Arg389 SNP. These kinetic differences correspond to significant variances in the response of β1AR to physiological calmodulin concentrations, peaking at ~50 nM. We also identified that β1AR‐calmodulin interactions are calcium‐dependent, with higher Ca2+ sensitivity of the Arg389 β1AR–CaM interaction over the entire physiological Ca2+ range. These results suggest that calmodulin binding to β1AR is important for its functions and our observed different biochemical properties of these interactions may explain the different responses in subjects carrying the Arg389 vs the Gly389 SNPs.Support or Funding InformationIOER grant 091707 to Q‐KT.

Mycorrhiza Regulates Signal Substance Levels and Pathogen Defense Gene Expression to Resist Citrus Canker

Citrus canker is a quarantined disease, severely harming citrus plants. Soil beneficial arbuscular mycorrhizal fungi (AMF) can provide a biological control pathway to resist pathogens. This work was to test changes of signal substances including hydrogen peroxide (H2O2), nitric oxide (NO), calmodulin (CaM), salicylic acid (SA) and jasmonic acid (JA) and the pathogen defense gene expression in roots of AMF (Paraglomus occultum) and non-AMF trifoliate orange (Poncirus trifoliata) seedlings after infected by a expressions citrus canker pathogen (Xanthomonas axonopodis pv. Citri, Xac). AMF inoculation significantly improved plant height, stem diameter and leaf number. Xac infection dramatically decreased root H2O2, NO, and SA levels, but increased root CaM and JA concentrations in non-AMF seedlings. There were higher H2O2 and CaM levels and lower JA levels in Xac-infected seedlings than in non-Xac-infected seedlings under mycorrhization. Under non-Xac infection, mycorrhizal treatment reduced root H2O2, NO, and SA but increased CaM and JA levels. However, under Xac infection, mycorrhizal inoculation distinctly accelerated root H2O2, NO, CaM, and SA accumulation, accompanied with up-regulated expression levels of root PtEPS1 (enhance pseudomonas susceptibility 1) and PtPR4 (pathogenesis related gene 4), indicating that Xac stimulated mycorrhizal roles in enhancing resistance of citrus canker. Such results imply that citrus plants with pre-inoculated AMF had stronger resistance to Xac infection through increasing signal substrate accumulation and pathogen defense gene expressions.
 
 *********
 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
 *********

Characterization of filarial phosphoglycerate kinase

Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45 kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca2+on the activity of rBmPGK was studied at different concentration of CaM (0.01–5.0 μM) and calcium chloride (0.01–100 μM). The rBmPGK was activated up to 85% in the presence of CaM at 1 μM and 10 μM concentration of CaCl2. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90 ± 10) % in ScPGK activity was observed in the presence of CaM and CaCl2 at 1.0 μM and 1.0 mM respectively, further increase in the conc. of CaCl2, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain.On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca2+-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kd = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

Responses of Four Citrus Plants to Phytophthora-Induced Root Rot

China is one of the largest citrus producers in Asia, where Phytophthora parasitica infection has become the major threat in sustaining long term citrus production. The proposed study examined the effects of P. parasitica on Citrus junos, C. limon, C. tangerina and Poncirus trifoliata to evaluate the resisted rootstock to Phytophthora root rot. P. parasitica infection notably decreased plant growth, root morphology and activities of pathogenesis-related proteins (PRs) in C. limon and C. tangerina. Root β-1,3-glucanase, chitinase and phenylalanine ammonialyase activities significantly increased in C. junos and P. trifoliata after infection with P. parasitica. P. parasitica infection notably decreased root salicylic acid concentrations in C. limon, C. tangerina and P. trifoliata, while increasing it in C. junos. An opposite trend was observed in root jasmonic acid levels after infection with P. parasitica, relative to root salicylic acid. Root nitric oxide and calmodulin concentrations were significantly increased in P. parasitica-infected C. junos, C. tangerina and P. trifoliata, while C. limon exhibited a decrease. These results demonstrated that citrus species like C. junos and P. trifoliata displayed a much higher resistance to Phytophthora-induced root rot, and C. limon and C. tangerina showed a comparatively lower degree of resistance.

Effects of deoxynivalenol exposure on cerebral lipid peroxidation, neurotransmitter and calcium homeostasis of chicks in vivo

During current research, the effects of deoxynivalenol (DON) exposure on cerebral lipid peroxidation, neurotransmitter secretion and calcium homeostasis in chicks were evaluated. One hundred and twenty Hailan chicks (male, 1-day-old) were randomly divided into four groups. Chicks in low, medium and high dose groups were fed with 0.27, 1.68 and 12.21 mg/kg−1 DON respectively by gavage according to feed intake. Chicks in control group were fed with physiological saline by gavage. The trials were conducted for 36 d. At the end of the trials, twenty chicks per group were sacrificed, and the cerebra were collected for measuring the brain indices. Compared with the control group, the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase were significantly decreased in treatment groups (P < 0.05), the contents of malondialdehyde in high dose group were increased (P < 0.05), the catalase activities and nitric oxide contents in medium and high dose groups were decreased (P < 0.05), and the activities of T-AOC in high dose group were reduced (P < 0.05). Compared with the control group, the concentrations of norepinephrine and 5-hydroxytryptamine in high dose group were obviously increased (P < 0.05), while the concentrations of dopamine were decreased (P < 0.05). Meanwhile, the concentrations of calcium and calmodulin (CaM) in medium and high dose groups were lower than those of the control group (P < 0.05), and the gene relative expression of CaM mRNA in treatment groups were significantly reduced (P < 0.05), in a dose-dependent manner. These results suggested that DON exposure can affect the cerebral lipid peroxidation, neurotransmitters secretion and the balance of calcium homeostasis in chicks.

Differential regulation of calmodulin, phenylalanine ammonia-lyase, and salicylic acid in response to Botrytis cinerea infection in tomato with different Ca2+ concentrations

ABSTRACTTo investigate the responses to Botrytis cinerea (B. cinerea) infection and the effects of exogenous Ca2+ on calmodulin (CaM), phenylalanine ammonia-lyase (PAL), and salicylic acid (SA) biosynthesis and concentrations, we investigated the role of exogenous calcium in defense and resistance to the necrotrophic fungus B. cinerea in tomato (Lycopersicon esculentum Mill.). Specifically, 7.5 mM Ca2+ was the best treatment for plant resistance, resulting in stable and permanent resistance to the pathogen. This treatment improved plant resistance and the results also confirmed that the concentrations of CaM and SA are negatively correlated with the resistance response.

The interactome and spatial redistribution feature of Ca2+ receptor protein calmodulin reveals a novel role in invadopodia-mediated invasion

Numerous studies have shown that calmodulin (CaM) is a major regulator of calcium-dependent signaling, which regulates cell proliferation, programmed cell death, and autophagy in cancer. However, limited information is available on mechanisms underlying the effect of CaM on the invasive property of glioblastoma multiforme (GBM) cells, especially with respect to invadopodia formation. In this study, we find that CaM serves as a prognostic factor for GBM, and it is strongly associated with the invasive nature of this tumor. Results of preliminary experiments indicated that CaM concentration was significantly correlated with the invasive capacity of and invadopodia formation by different GBM cell lines. CaM inhibition via a small hairpin RNA or a pharmacological inhibitor significantly disrupted invadopodia formation and MMP activity and downregulated vimentin expression. Moreover, CaM knockdown exerted a strong anti-invasive effect on GBM in vivo. Interestingly, epidermal growth factor treatment promoted CaM redistribution from the nucleus to the cytoplasm, eventually activating invadopodia-associated proteins by binding to them via their cytosolic-binding sites. Moreover, CaM inhibition suppressed the activation of invadopodia-associated proteins. Thus, our findings provide a novel therapeutic strategy to impede GBM invasion by inhibiting invadopodia formation, and shed light on the spatial organization of CaM signals during GBM invasion.

Abstract B048: Activation of PKC inhibits oncogenic KRAS-driven malignancies

Abstract The extremely high mutation rate of KRAS in three of the top four deadly cancers (lung, colon, and pancreas) makes it a major target in anticancer drug discovery. However, no effective anti-KRAS inhibitors have yet been developed into clinically useful drugs. Therefore, identifying “druggable” signaling components crucial for KRAS can provide a new approach for targeting KRAS-driven malignancies. We have reported that oncogenic KRAS uniquely elicits a tumorigenic phenotype through downregulation of noncanonical Fzd8/Wnt/Ca2+ signaling. Binding of calmodulin (CaM) to KRAS, but not to H- or N-RAS, appears to be responsible for this major difference. The total intracellular concentration of CaM has been reported to be significantly below the total concentration of its targets, making CaM a limiting factor in their regulation. Therefore, the unique binding of KRAS to CaM, which competes for a limiting pool of CaM, can be sufficient to affect CaM-dependent signaling pathways. Serine-181 in the hypervariable region of KRAS, as the essential domain for KRAS to bind to CaM, has been identified as the phosphorylation site of PKC. The KRAS-V12-S181D (pseudo-phosphorylated) variant lost the ability to suppress CaMKii activity and Fzd8 expression, suggesting that the interaction between KRAS and CaM is an important pathway for KRAS to inhibit Fzd8-mediated noncanonical Wnt/Ca2+ signaling. Thus, we hypothesize to target KRAS selectively by blocking this specific interaction between KRAS and CaM through the activation of PKC. The activation of PKC by a non-tumor promoting PKC activator, prostratin, successfully interrupts the KRAS-CaM interaction, rewires the sequential downstream signaling pathways, and, most importantly, represses the tumor initiation and growth in KRAS-driven cancers. The pancreatic cancer cells treated with Gο6984 (targeting conventional and novel PKCs), but not Gο6976 (targeting conventional PKCs), lost the response to prostratin treatment on activation of CaMKii. Next, each individual of PKC isozyme in conventional and novel subfamilies has been knocked down by siRNA, followed by the treatment of prostratin. The cells, where PKCδ has been knocked down, failed to activate CaMKii in the response to prostratin, when compared to the cells with the depletions of other PKC isoforms. The pancreatic cancer cells where PKCδ, but not PKCα, has been knocked down by shRNA lost their sensitivity to prostratin on cell viability and growth in vitro. The treatment of prostratin increased the phosphorylated PKCδ at serine-299 in KRAS transformed cells, suggesting prostratin acts as a PKCδ activator. The cellular thermal shift assay suggested that, when compared to PMA, prostratin preferentially stabilized PKCδ in a dose-dependent manner. To further define the specificity of prostratin-induced PKC activation on phosphorylation of KRAS, we treated the LSL-KRAS-G12D mice carrying the additional mutation at serine-181 (S181S to A, resistant to phosphorylation) with prostratin post-adenoCre infection. The lung tumors from LSL-KRAS-G12D-S181S mice, but not -S181A ones, showed increased phosphorylated CaMKii in the response to prostratin treatment. The results suggest that activation of certain specific PKC isozymes may provide a novel approach to rewire KRAS-mediated signaling pathway and to sequentially suppress its malignant features in human cancers. Citation Format: Man-Tzu Wang, Jacqueline Galeas, Frank McCormick. Activation of PKC inhibits oncogenic KRAS-driven malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B048.

Toxic effects of Pb2+ entering sperm through Ca2+ channels in the freshwater crab Sinopotamon henanense

Lead (Pb) is a heavy metal that can damage animal sperm. To study the effects of Pb on calcium homeostasis and calcium channel in the sperm of freshwater crab Sinopotamon henanense, the induction of acrosome reaction (AR) and acrosin activity were investigated when crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L) for 3, 5 and 7 d separately. Fluorescent probe Fluo-3/AM was loaded into the sperm, and [Ca2+] in the sperm was measured by fluorescence microscopy and using microplate reader. The calmodulin (CaM) concentration was measured by ELISA method. Verapamil (VRP), a calcium channel blocker, was used to evaluate whether Pb can enter the sperm through calcium channels leading to sperm damage. After sperm were exposed at 50μg/L VRP, 100μg/L Pb, 50μg/L VRP+100μg/L Pb, 1000μg/L Pb and 50μg/L VRP+1000μg/L Pb for 1h in vitro，sperm quality parameters (sperm survival and sperm DNA integrity) and levels of parameters indicating oxidative stress (protein carbonylation [PCO] and malondialdehyde [MDA]) were measured. Our data showed that Pb reduced the induction of acrosome reaction (AR), down-regulated the acrosin activity, decreased the intracellular concentration of Ca2+ and elevated CaM concentration. Compared to controls, Pb alone induced significant stress, as reflected by decreasing sperm survival and sperm DNA integrity, and increasing PCO and MDA contents. In the presence of VRP, 100μg/L Pb-induced stresses were reduced, all the measured parameters in the sperm exposed at 100μg/L Pb returned to control levels. Our results indicate that Pb enters the sperm of the crab S. henanense through calcium channels, the inhibition of which blocks Pb-induced stresses such as sperm quality decline and oxidative damage.

Mycorrhizal Fungi Regulate Root Responses and Leaf Physiological Activities in Trifoliate Orange

Plant responses to mycorrhization are mediated through secretion of certain signal molecules deposited in mycorrhizosphere in response to environmental stimuli. Responses of four arbuscular mycorrhizal fungi (AMF), namely Claroideoglomus etunicatum, Diversispora versiformis, Funneliformis mosseae, and Rhizoglomus intraradices on root morphology, lateral root (LR) number, and leaf carbohydrates, nitric oxide (NO), and calmodulin (CaM) changes were studied using trifoliate orange. Inoculation response of D. versiformis, F. mosseae, and R. intraradices registered significantly higher plant growth performance (plant height, stem diameter, leaf number, and shoot and root biomass), root morphological traits (total length, projected area, surface area, and volume), and LR number (first-, second-, third-, and forth-order), compared to un-inoculated response. Higher concentrations of CaM, NO, glucose, and fructose and lower sucrose level in leaves were observed in AMF-seedlings than in non-AMF seedlings. Correlation studies further revealed, root morphological traits and LR numbers were significantly negatively correlated with sucrose whereas positively correlated with glucose, fructose, NO, and CaM level in leaves. These results suggested, AMF-induced root modification is routed through sucrose cleavage and partly through changes in NO and CaM.

Characterization of the Orai-Calmodulin Interaction as Potential Mediator of Calcium-Dependent Orai-Channel Inactivation

Calcium is an important second messenger involved in many cellular processes such as cell proliferation, T-cell activation, muscle contraction, egg fertilization, or apoptosis. Calcium entry into non-excitable cells is mainly carried by store-operated Ca2+ release-activated Ca2+ (CRAC) channels, whereby Orai was found to be the calcium channel in the plasma membrane and the stromal interaction molecule (STIM) to act as a calcium sensor in the endoplasmatic reticulum (ER), and as an activator of Orai channels. A reduction of Ca2+ in the ER causes STIM to oligomerize, enabling its accumulation at the ER-plasma membrane junctions where it binds directly to Orai to open the Ca2+-channel. Compared with activation, much less is known about the mechanisms underlying Ca2+-dependent inactivation (CDI) processes. It has been proposed that binding of calmodulin (CaM) to a highly conserved N-terminal segment of Orai1 adjacent to its first transmembrane helix is critical for CDI. Hereby it is assumed that CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation. However, the molecular basis of these interactions remains unclear. In this study, the interaction between calmodulin and the N-terminal fragment of Orai1 and its homologue Orai3 was studied by Single Molecule Recognition Force Spectroscopy, Surface Plasmon Resonance, and Microscale Thermophoresis. Both Orai fragments showed a calcium-dependent and highly specific interaction with calmodulin, with slightly longer bond lifetimes and lower dissociation rate constants for Orai3 than for Orai1. Our data support a switch between a 1:1 and a 1:2 binding stoichiometry between CaM and Orai1/3. The latter occurs at low CaM concentration, has much higher affinity, and takes much longer to disscociate after removal of Ca2+ by EGTA. By comparison, only 1:1 binding with high affinity was found between CaM and smooth muscle myosin light chain kinase. This emphasizes the importance of the unique amino acid sequence of the conserved N-terminal Orai segment for high CaM affinity and CDI. This work was supported by the state of Upper Austria (project DK NanoCell).