Calcium-dependent Activity Research Articles

Fatty acids influence the efficacy of lutein in the modulation of α-crystallin chaperone function: Evidence from selenite induced cataract rat model

BackgroundLoss of α-crystallin chaperone function results in the lens protein aggregation leading to cataract. In this study, we evaluated the efficacy of micellar lutein with different fatty acids in modulating α-crystallin chaperone function under selenite cataract conditions. MethodsCataract was induced in rat pups by giving sodium selenite (25 μM/kg body weight) by IP. Lutein [(L), 1.3 μmol/kg body weight)] was given day before and five days after selenite injection as a micelle with 7.5 mM linoleic acid (LA), or 7.5 mM eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) or 7.5 mM oleic acid (OA). Lens α-crystallins was purified, and its chaperone function and integrity was assessed. Cholesterol, calcium, calpain-2, procaspase-3, and expression of α-A and β-B1 crystallin in the lens of cataract and micellar lutein administered rats were evaluated. ResultsCataract induction significantly (p < 0.05) decreased lens α-crystallin chaperone function. Cataract rats had increased cholesterol and calcium level, increased the expression of calpain-2, and α-A and β-B1 crystallin, and reduced the pro-caspase-3 level in the lens. However, micellar lutein administration significantly (p < 0.05) protected client proteins from aggregation via the modulation of calcium-dependent calpain-2 protease activity. The chaperone function of lens α-crystallins in rats administered micellar lutein with EPA + DHA was found to be highest when compared to OA and LA. ConclusionsMicellar lutein with unsaturated fatty acids beneficially modulates α-crystallin chaperone function. Among the fatty acids tested, micellar lutein with EPA + DHA exhibited superior effects, thereby offering a promising strategy for cataract management.

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin

Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting ‘AC-to-Hly-linking segment’ (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411–490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411–490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA411–490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the ‘AC-to-Hly-linking segment’ (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

Microplastic exposure represses the growth of endosymbiotic dinoflagellate Cladocopium goreaui in culture through affecting its apoptosis and metabolism.

Microplastics are widespread emerging marine pollutants that have been found in the coral reef ecosystem. In the present study, using Cladocopium goreaui as a symbiont representative, we investigated cytological, physiological, and molecular responses of a Symbiodiniaceae species to weeklong microplastic exposure (Polystyrene, diameter 1.0μm, 9.0×109 particles L-1). The density and size of algal cells decreased significantly at 7d and 6-7d of microplastic exposure, respectively. Chlorophyll a content increased significantly at 7d of exposure, whereas Fv/Fm did not change significantly during the entire exposure period. We observed significant increases in superoxide dismutase activity and caspase3 activation level, significant decrease in glutathione S-transferase activity, but no change in catalase activity during the whole exposure period. Transcriptomic analysis revealed 191 significantly upregulated and 71 significantly downregulated genes at 7d after microplastic exposure. Fifteen GO terms were overrepresented for these significantly upregulated genes, which were grouped into four categories including transmembrane ion transport, substrate-specific transmembrane transporter activity, calcium ion binding, and calcium-dependent cysteine-type endopeptidase activity. Thirteen of the significantly upregulated genes encode metal ion transporter and ammonium transporter, and five light-harvesting protein genes were among the significantly downregulated genes. These results demonstrate that microplastics can act as an exogenous stressor, suppress detoxification activity, nutrient uptake, and photosynthesis, elevate oxidative stress, and raise the apoptosis level through upregulating ion transport and apoptotic enzymes to repress the growth of C.goreaui. These effects have implications in negative impacts of microplastics on coral-Symbiodiniaceae symbiosis that involves C.goreaui.

Neurotoxicity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom in vertebrate neuromuscular preparations in vitro and neutralization by antivenom.

We investigated the effect of South American coralsnake (Micrurus lemniscatus lemniscatus) venom on neurotransmission in vertebrate nerve-muscle preparations in vitro. The venom (0.1-30µg/ml) showed calcium-dependent PLA2 activity and caused irreversible neuromuscular blockade in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, contractures to exogenous acetylcholine and carbachol (CCh), but not KCl, were abolished by venom concentrations ≥ 0.3µg/ml; in PND preparations, the amplitude of the tetanic response was progressively attenuated, but with little tetanic fade. In low Ca2+ physiological solution, venom (10µg/ml) caused neuromuscular blockade in PND preparations within ~ 10min that was reversible by washing; the addition of Ca2+ immediately after the blockade temporarily restored the twitch responses, but did not prevent the progression to irreversible blockade. Venom (10µg/ml) did not depolarize diaphragm muscle, prevent depolarization by CCh, or cause muscle contracture or histological damage. Venom (3µg/ml) had a biphasic effect on the frequency of miniature end-plate potentials, but did not affect their amplitude; there was a progressive decrease in the amplitude of evoked end-plate potentials. The amplitude of compound action potentials in mouse sciatic nerve was unaffected by venom (10µg/ml). Pre-incubation of venom with coralsnake antivenom (Instituto Butantan) at the recommended antivenom:venom ratio did not neutralize the neuromuscular blockade in PND preparations, but total neutralization was achieved with a tenfold greater volume of antivenom. The addition of antivenom after 50% and 80% blockade restored the twitch responses. These results show that M. lemniscatus lemniscatus venom causes potent, irreversible neuromuscular blockade, without myonecrosis. This blockade is apparently mediated by pre- and postsynaptic neurotoxins and can be reversed by coralsnake antivenom.

Induction of calcium-dependent protein kinase activity and HmCDPK1 expression in the early response of Hami melons to Penicillium infection

Calcium-dependent protein kinase (CDPK) plays an important role in plant resistance to disease. In this study, we assessed CDPK activity in Hami melons during Penicillium infection, in order to investigate the possible role of CDPK in this context. The results showed the induction of CDPK by Penicillium infection may contribute to the early stages of disease resistance in Hami melons. In order to further study the molecular mechanisms underlying this response, relatively high expression of HmCDPK1 was screened form the transcriptome database of Hami melons. Bioinformatics analysis showed HmCDPK1 had a length of 2365 bp, with a maximum open reading frame of 1560 nucleotides, encoding 519 amino acids with a molecular weight of 58,646.83. Moreover, its theoretical isoelectric point was 6.34, as a hydrophilic and non-transmembrane protein. Real-time quantitative PCR showed the transcript of HmCDPK1 was persistently increased after Penicillium infection and reached its maximum at 12 h, being significantly higher than in non-infected plants. These results suggest the induction of CDPK activity and HmCDPK1 expression in the early response of Hami melons to Penicillium may contribute to the resistance against this infection.

Proteomic Analysis of Plasmodesmata From Populus Cell Suspension Cultures in Relation With Callose Biosynthesis.

Plasmodesmata are channels that link adjacent cells in plant tissues through which molecular exchanges take place. They are involved in multiple processes vital to plant cells, such as responses to hormonal signaling or environmental challenges including osmotic stress, wounding and pathogen attack. Despite the importance of plasmodesmata, their proteome is not well-defined. Here, we have isolated fractions enriched in plasmodesmata from cell suspension cultures of Populus trichocarpa and identified 201 proteins that are enriched in these fractions, thereby providing further insight on the multiple functions of plasmodesmata. Proteomics analysis revealed an enrichment of proteins specifically involved in responses to stress, transport, metabolism and signal transduction. Consistent with the role of callose deposition and turnover in the closure and aperture of the plasmodesmata and our proteomic analysis, we demonstrate the enrichment of callose synthase activity in the plasmodesmata represented by several gene products. A new form of calcium-independent callose synthase activity was detected, in addition to the typical calcium-dependent enzyme activity, suggesting a role of calcium in the regulation of plasmodesmata through two forms of callose synthase activities. Our report provides the first proteomic investigation of the plasmodesmata from a tree species and the direct biochemical evidence for the occurrence of several forms of active callose synthases in these structures. Data are available via ProteomeXchange with identifier PXD010692.

Daptomycin-loaded biodegradable thermosensitive hydrogels enhance drug stability and foster bactericidal activity against Staphylococcus aureus

A drug delivery system based on fully biodegradable thermosensitive hydrogels enabling controlled antibiotic release may support the management of implant-associated infections. In this work, the lipopeptide antibiotic daptomycin was encapsulated in hydrogel networks consisting of vinyl sulfonated triblock copolymers of PEG-p(HPMAm-lac1,2) and thiolated hyaluronic acid. High concentrations of active daptomycin exceeding the minimum biofilm eradicating concentration were sustainably eluted from the biodegradable carrier. The drug release profiles were tailored by varying the degree of substitution (DS) of thiol groups of hyaluronic acid, reaching a plateau level after 200 and 330 h for DS values of 53% and 31%, respectively. The hydrogel polymeric network preserved the structural stability of the loaded antibiotic and retained the calcium-dependent daptomycin activity, showing a noticeable biofilm bactericidal effect against a 24 h-old Staphylococcus aureus biofilm in vitro. The two-component thermosensitive hydrogels demonstrated to be an excellent antibiotic releasing scaffold with potential clinical applications in the management of implant-associated infections.

Plasmodium falciparum histidine triad protein and calmodulin modulates calcium homeostasis and intracellular proteolysis

Calcium signaling has an essential role in fundamental processes of Plasmodium life cycle, including migration, cell invasion and parasite development. Two important players in calcium homeostasis, the Histidine Triad (HIT) protein that is implicated in calcium signaling in mammalian cells and calmodulin, which is a classic calcium sensor in eukaryotes are present in Plasmodium falciparum, however theirs function is unknown in the parasite. Here, we investigated the involvement of the P. falciparum Histidine Triad protein (PfHint-1) and calmodulin (PfCaM) in calcium signaling and intracellular proteolysis. For this, we targeted PfHint-1 with a hemagglutinin tail and overexpressed both proteins. We observed that PfHint-1 is expressed throughout the erythrocytic stages and partially colocalizes to the endoplasmic reticulum. Parasites overexpressing PfHint-1 displayed lower ER Ca2+ content and a higher [Ca2+]cyt rise in the parasite cytosol upon Ca2+ addition to the extracellular medium after depletion of ER calcium store. PfCaM-overexpressing parasites exhibit a higher [Ca2+]cyt rise after challenge with the calmodulin inhibitor, calmidazolium. The calcium-dependent proteolytic activity in PfCaM- and PfHint-1-overexpressing parasites was increased and correlated to alterations in calcium homeostasis. Taken together, our results indicate the participation of these proteins in P. falciparum fundamental cellular processes and highlights promising targets for the development of antimalarial drugs.

EphA2 stimulates VCAM-1 expression through calcium-dependent NFAT1 activity

Endothelial cell activation by proinflammatory stimuli drives leukocyte recruitment through enhanced expression of counter-receptors such as vascular cell adhesion molecule-1 (VCAM-1). We previously demonstrated that activation of the receptor tyrosine kinase EphA2 with its ligand ephrin-A1 induces VCAM-1 expression. Here, we sought to characterize the proinflammatory signaling pathways involved. Analysis of over-represented transcription factors in ephrin-A1-induced genes identified multiple potential transcriptional regulators, including the Rel family members nuclear factor-κB (NF-κB/p65) and nuclear factor of activated T-cells (NFAT). While ephrin-A1 failed to induce endothelial NF-κB activation, NF-κB inhibitors prevented ephrin-A1-induced VCAM-1 expression, suggesting basal NF-κB activity is required. In contrast, ephrin-A1 induced a robust EphA2-dependent increase in NFAT activation, and mutation of the NF-κB/NFAT-binding sites in the VCAM-1 promoter blunted ephrin-A1-induced promoter activity. NFAT activation classically occurs through calcium-dependent calcineurin activation, and inhibiting NFAT signaling with calcineurin inhibitors (cyclosporine A, FK506) or direct NFAT inhibitors (A-285222) was sufficient to block ephrin-A1-induced VCAM-1 expression. Consistent with robust NFAT activation, ephrin-A1-induced an EphA2-dependent calcium influx in endothelial cells that was required for ephrin—A1-induced NFAT activation and VCAM-1 expression. This work provides the first data showing EphA2-dependent calcium influx and NFAT activation and identifies NFAT as a novel EphA2-dependent proinflammatory pathway in endothelial activation.

Alternative RNA splicing of leucocyte tissue transglutaminase in coeliac disease

Tissue transglutaminase is a ubiquitous and multifunctional protein that contributes to several processes such as apoptosis/survival, efferocytosis, inflammation and tissue repairing under physiological and pathological conditions. Several activities can be associated with well-established functional domains; in addition, four RNA alternative splice variants have been described, characterized by sequence divergences and residues deletion at the C-terminal domains. Tissue transglutaminase is recognized as the central player in the physiopathology of coeliac disease (CD) mainly through calcium-dependent enzymatic activities. It can be hypothesized that differential regulation of tissue transglutaminase splice variants expression in persons with CD contributes to pathology by altering the protein functionality. We characterized the expression pattern of RNA alternative splice variants by RT-PCR in peripheral cells from patients with CD under free gluten diet adhesion; we considered inflammatory parameters and specific antibodies as markers of the stage of disease. We found significant higher expression of both the full length and the shortest C-truncated splice variants in leucocytes from patients with CD in comparison with healthy individuals. As tissue transglutaminase expression and canonical enzymatic activity are linked to inflammation, we studied the RNA expression of inflammatory cytokines in peripheral leucocytes of persons with CD in relation with splice variants expression; interestingly, we found that recently diagnosed patients showed significant correlation between both the full length and the shortest alternative spliced variants with IL-1 expression. Our results points that regulation of alternative splicing of tissue transglutaminase could account for the complex physiopathology of CD.

Plasma Lipid Metabolism is altered in Acute Mild Traumatic Brain Injury

Background and ObjectivesLipids are the source of signaling and inflammatory molecules that may contribute to trauma pathology. Accordingly, we examined levels of lipid species and a lipolytic enzyme activity in a one‐month longitudinal study of acute mild traumatic brain injury (mTBI).MethodsWe recruited mTBI patients (n=21) and non‐head trauma controls (CT, n=9), aged 18–50 years from the emergency department of the Huntington Memorial Hospital (Pasadena, CA). Symptom progression was observed within days of injury (W1), and four weeks thereafter (W4). Plasma lipids collected at W1 and W4 were extracted and unesterified (UFA) and esterified fatty acids (EFA) quantified using negative ion/chemical ionization gas chromatography/mass spectrometry. Plasma glycerophospholipids (GP) and sphingolipids (SP) were analyzed using liquid chromatography‐tandem mass spectrometry. Plasma phospholipase A2 (PLA2) activity was determined using a fluorescence‐based activity assay.ResultsmTBI participants' plasma at W1 had higher levels of 24 UFA species that included seven saturated fatty acids (SAFA), seven monounsaturated fatty acids (MUFA), four omega‐3, and six omega‐6 polyunsaturated fatty acids (PUFA). The sum of all SAFAs, MUFAs, omega‐3 PUFAs, omega‐6 PUFAs, and PUFAs were higher in mTBI plasma at W1. For EFA at W1, two omega‐6 (homo‐g‐C20:3n‐6, C22:4n‐6), one omega‐3 (C20:5n‐3), the sum of omega‐3 PUFAs, and the omega‐3 to omega‐6 ratios (omega‐3 index) were lower in mTBI than in CT. At W4, esterified C19:0 and C22:3n‐3 levels were lower while C24:1 was higher in mTBI compared with CT. The UFA to EFA ratio that estimates endogenous lipolysis of fatty acids was higher in mTBI than CT for three SAFAs, two MUFAs, two omega‐6, two omega‐3, and the ratio of the sum of UFAs to the sum EFAs. Calcium‐dependent PLA2 activity was higher in mTBI plasma at W1 but not at W4. mTBI PLA2 activity at W1 positively correlated with most UFA species except C18:2n‐6 and C18:3n‐6 but correlated only with two UFA (C20:1, C22:1) in CT. PLA2 activity negatively correlated with seven EFA species.ConclusionsOur data showing higher UFA, UFA to EFA ratios, and correlation of UFA with PLA2 indicate excessive lipolysis in early mTBI. A lower omega‐3 index suggests the excessive oxidative breakdown of omega‐3 in mTBI. We propose that early intervention using strategies that reduce lipolysis may attenuate tissue damage linked to mTBI. Additionally, measurements of fatty acid fluctuations may be useful in discovering new therapies and monitoring resolution of mTBI.Support or Funding InformationFinancial support provided by the D.O.D. grant W81XWH‐13‐1‐0005. Thao Tran, David Strickland, Betty Chung, Darlene Royal, and David Buennagel assisted with data collection and patient recruitment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New insight into transglutaminase 2 and link to neurodegenerative diseases.

Formation of toxic protein aggregates is a common feature and mainly contributes to the pathogenesis of neurodegenerative diseases (NDDs), which include amyotrophic lateral sclerosis (ALS), Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. The transglutaminase 2 (TG2) gene encodes a multifunctional enzyme, displaying four types of activity, such as transamidation, GTPase, protein disulfide isomerase, and protein kinase activities. Many studies demonstrated that the calcium-dependent transamidation activity of TG2 affects the formation of insoluble and toxic amyloid aggregates that mainly consisted of NDD-related proteins. So far, many important and NDD-related substrates of TG2 have been identified, including amlyoid-β, tau, α-synuclein, mutant huntingtin, and ALS-linked trans-activation response (TAR) DNA-binding protein 43. Recently, the formation of toxic inclusions mediated by several TG2 substrates were efficiently inhibited by TG2 inhibitors. Therefore, the development of highly specific TG2 inhibitors would be an important tool in alleviating the progression of TG2-related brain disorders. In this review, the authors discuss recent advances in TG2 biochemistry, several mechanisms of molecular regulation and pleotropic signaling functions, and the presumed role of TG2 in the progression of many NDDs.

Synthesis and evaluation of analogues of the glycinocin family of calcium-dependent antibiotics.

The glycinocins are a class of calcium-dependent, acidic cyclolipopeptide antibiotics that are structurally related to the clinically approved antibiotic daptomycin. In this article, we describe the synthesis of a small library of glycinocin analogues that differ by variation in the exocyclic fatty acyl substituent. The glycinocin analogues were screened against a panel of Gram-positive bacteria (as well as Gram-negative P. aeruginosa). These analogues exhibited similar calcium-dependent activity to the parent natural products against Gram-positive bacteria but showed no activity against P. aeruginosa. The length of the fatty acid was shown to be important for optimal biological activity, while the hybridisation at the α,β position and branching within the fatty acyl chain had only subtle effects on activity.

Total Synthesis of Glycinocins A-C.

The glycinocins are a class of calcium-dependent, acidic cyclolipopeptide antibiotics structurally related to the clinically approved daptomycin. Herein, we describe a divergent total synthesis of glycinocins A-C, which differ in the structure of a branched α,β-unsaturated fatty acyl moiety. The three natural products exhibited calcium-dependent antimicrobial activity against Staphylococcus aureus and Bacillus subtilis with MICs ranging from 5.5 to 17 μM.

In vitroselection ofPhytomonas serpenscells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Structure-Activity Relationships of Potent, Targeted Covalent Inhibitors That Abolish Both the Transamidation and GTP Binding Activities of Human Tissue Transglutaminase.

Human tissue transglutaminase (hTG2) is a multifunctional enzyme. It is primarily known for its calcium-dependent transamidation activity that leads to formation of an isopeptide bond between glutamine and lysine residues found on the surface of proteins, but it is also a GTP binding protein. Overexpression and unregulated hTG2 activity have been associated with numerous human diseases, including cancer stem cell survival and metastatic phenotype. Herein, we present a series of targeted covalent inhibitors (TCIs) based on our previously reported Cbz-Lys scaffold. From this structure-activity relationship (SAR) study, novel irreversible inhibitors were identified that block the transamidation activity of hTG2 and allosterically abolish its GTP binding ability with a high degree of selectivity and efficiency (kinact/KI > 105 M-1 min-1). One optimized inhibitor (VA4) was also shown to inhibit epidermal cancer stem cell invasion with an EC50 of 3.9 μM, representing a significant improvement over our previously reported "hit" NC9.

Phosphatidylserine: A cancer cell targeting biomarker.

Cancer is a leading cause of mortality and morbidity globally. Many prominent cancer-associated molecules have been identified over the recent years which include EGFR, CD44, TGFbRII, HER2, miR-497, NMP22, BTA, Fibrin/FDP etc. These biomarkers are often used for screening, detection, diagnosis, prognosis, prediction and monitoring of cancer development. Phosphatidylserine (PS) is an essential component in all human cells which is present on the inner leaflet of the cell membrane. The oxidative stress causes exposure of PS on the surface of the vascular endothelium in the cancer cells (lung, breast, pancreatic, bladder, skin, brain metastasis, rectal adenocarcinoma etc.) but not on the normal cells. The external PS is regulated by calcium-dependent flippase activity. Cancer cell lines with high surface PS have low flippase activity and high intracellular calcium content. Human Annexin-V, PS targeting antibodies (PGN635 and bavituximab and mch1N11), lysosomal protein, phospholipid Saposin C dioleoylphosphatidylserine (SapC-DOPS), peptide-peptoid hybrid PPS1, PS-binding 14-mer peptide (PSBP-6) and hexapeptide (E3) have been reported to target PS present on cancer cell surface. High expression of CD47 inhibits tumor cell phagocytosis by macrophages. The PS cancer biomarker has also been used to target the drugs to cancer cells specifically without affecting other healthy cells. Currently, the fusion protein (FP) consisting of L-methionase linked to human Annexin-V has been reported to target the cancer cells. The FP catalyzes the conversion of non-toxic prodrug selenomethionine into toxic methyl selenol which thus also prevents the methionine (essential amino acid) supplementation to the cancer cells.

The actin binding protein scinderin acts in PC12 cells to tether dense-core vesicles prior to secretion.

Mechanistic understanding of the control of vesicle motion from within a secretory cell to the site of exocytosis remains incomplete. In this work, we have used total internal reflection (TIRF) microscopy to examine the mobility of secretory vesicles at the plasma membrane. Under resting conditions, we found vesicles showed little lateral mobility. Anchoring of vesicles in this membrane proximal compartment could be disrupted with latrunculin A, indicating an apparent actin dependent process. A candidate intermediary between vesicles and the actin skeleton is the actin binding protein scinderin. Co-transfection of an shRNA construct against scinderin blocked secretion, and also increased the mobility of vesicles in the membrane-proximal section of the cell, indicating a dual role for scinderin in secretion; tethering vesicles to the cytoskeleton, as well as liberating them following stimulation through the previously described calcium dependent actin severing activity. Analysis of lipid dependence indicates that scinderin exhibits calcium dependent binding to phosphatidyl-inositol monophosphate, providing a possible mechanism for vesicle binding.

Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase

BackgroundA 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. MethodsA soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. ResultsHere we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca2+-dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. General significanceThe study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general.

Purification and characterization of Cc-Lec, C-type lactose-binding lectin: A platelet aggregation and blood-clotting inhibitor from Cerastes cerastes venom

In this study, we reported for the first time the biochemical and structural characterization of Cc-Lec, a C-type lectin purified from Cerastes cerastes venom by affinity chromatography. This lectin was homogeneous by SDS-PAGE, and was shown to be a 34 271.59Da polypeptide by Electrospray mass spectrometry MS-ES-TOF. Its identified sequence of 160 amino acids corresponding to one subunit, revealed a high identity with other related proteins. Cc-Lec modeled 3D structure appeared as homodimer cross-linked by one disulfide bridge. Cc-Lec exhibited a calcium dependent hemagglutinating activity against human group O erythrocytes. Cc-Lec inhibited platelet aggregation induced by ADP, arachidonic acid or fibrinogen suggesting its interaction with their specific receptors namely P2Y1 and/or P2Y12, GPIIb/IIIa and TPα respectively. Cc-Lec was not lethal for mice until 10mg/kg administered by i.p. route. The lectin displayed a lasting anticoagulation on mice plasma even two days post-injection. This anticoagulation seems to be related to its interaction with coagulation factors Xa and IXa. Therefore, Cc-Lec prevented FXa amidolytic activity with Km=4.3310−4μg/mL and ki=14.4μg/mL. It seems to interact with these targets through CRD domain which could make it a good target as a pharmacological promising molecule in thrombosis diagnosis and therapy.