Calcium Binding Properties Research Articles

Acquired pellicle and biofilm engineering with CaneCPI-5: Insights from proteomic and microbiomics analysis

ObjectiveIn this in vivo proof-of-concept study, acquired pellicle engineering was implemented to promote alterations in the protein composition of the acquired enamel pellicle (AEP) and the bacterial composition of the dental biofilm after treatment with Sugarcane cystatin (CaneCPI-5). DesignAfter prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with the following solutions: 1) deionized water (H2O- negative control or 2) 0.1 mg/mL CaneCPI-5. The AEP and biofilm were formed along 2 or 3 h, respectively. The AEP was collected with electrode filter papers soaked in 3 % citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. The biofilm microbiome was collected with a dental curette. The DNA was extracted, amplified, and analyzed by 16S-rRNA Next Generation Sequencing (NGS). ResultsTreatment with CaneCPI-5 increased several proteins with antimicrobial, acid-resistance, affinity for hydroxyapatite, structural and calcium binding properties, such as Cysteine-rich-3 (6-fold-p = 0.03), Cystatin-B (5.5-fold-p < 0.01), Neutrophil-defensin 1 (4.7-fold-p < 0.01), Mucin (3.9-fold-p < 0.01), Immunoglobulin-heavy-constant (3.8-fold-p < 0.01) and Lactotransferrin (2.8-fold-p < 0.01). Microbiome revealed that several commensal bacteria had their abundance increased after rinsing with CaneCPI-5, such as Corynebacterium and Neisseria, while Streptococcus and Prevotella nigrescens were decreased. The results indicate the efficiency of CaneCPI-5 in promoting beneficial changes in the AEP and biofilm, making this phytocystatin a potential target for incorporation into dental products. ConclusionCane demonstrated the capability to alter the protein composition of the acquired enamel pellicle (AEP) and the initial colonizers of the biofilm, enhancing the presence of proteins and bacteria crucial for dental protection.

Characterization and Mechanism of a Novel Rice Protein Peptide (AHVGMSGEEPE) Calcium Chelate in Enhancing Calcium Absorption in Caco-2 Cells.

Rice protein peptides (RPP) are a potentially valuable source of high-quality calcium chelating properties. However, there is a lack of information regarding the calcium-absorption-promoting effect of RPP and its underlying mechanism. The present study adopted molecular docking methodologies to analyze the 10 most potent peptide segments from RPP. Results revealed that the peptide AHVGMSGEEPE (AHV) displayed optimal calcium binding properties (calcium-chelating capacity 55.69 ± 0.66 mg/g). Quantum chemistry analysis revealed that the AHV peptide effectively binds and forms stable complexes with calcium via the carbonyl oxygen atoms in valine at position 3 and the carbonyl of the C-terminal carboxyl group of glutamate at position 11. The spectral analysis results indicated that AHV may bind to calcium through carboxyl oxygen atoms, resulting in a transition from a smooth surface block-like structure to a dense granular structure. Furthermore, this study demonstrated that the 4 mmol/L AHV-Ca chelate (61.75 ± 13.23 μg/well) significantly increases calcium absorption compared to 1 mM CaCl2 (28.57 ± 8.59 μg/well) in the Caco-2 cell monolayer. In terms of mechanisms, the novel peptide-calcium chelate AHV-Ca derived from RPP exerts a cell-level effect by upregulating the expression of TRPV6 calcium-ion-channel-related genes and proteins (TRPV6 and Calbindin-D9k). This study provides a theoretical basis for developing functional foods with the AHV peptide as ingredients to improve calcium absorption.

Acquired Pellicle and Biofilm Engineering by Rinsing with Hemoglobin Solution

Introduction: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the “acquired pellicle engineering” concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization. Objective: Combining in vivo and in vitro proof-of-concept protocols, we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization. Methods: In the in vivo studies, 10 volunteers, in 2 independent experiments, rinsed (10 mL,1 min) with deionized water-negative control or 1.0 mg/mL Hb. The AEP and biofilm formed along 2 or 3 h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA next-generation sequencing (NGS). In in vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with (1) phosphate-buffered solution (PBS), (2) 0.12% chlorhexidine, (3) 500 ppm NaF, (4) 1.0 mg/mL Hb, (5) 2.0 mg/mL Hb, and (6) 4.0 mg/mL Hb. The biofilm was cultivated for 5 days. Resazurin, colony forming units (CFU), and transversal microradiography were performed. Results: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU, and enamel demineralization compared to PBS. Conclusion: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.

The Dictyostelium discoideum FimA protein, unlike yeast and plant fimbrins, is regulated by calcium similar to mammalian plastins

Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory ‘switch helix’ that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.

Calcium-binding properties, stability, and osteogenic ability of phosphorylated soy peptide-calcium chelate.

Bioactive peptides based on foodstuffs are of particular interest as carriers for calcium delivery due to their safety and high activity. The phosphorylated peptide has been shown to enhance calcium absorption and bone formation. A novel complex of peptide phosphorylation modification derived from soybean protein was introduced, and the mechanism, stability, and osteogenic differentiation bioactivity of the peptide with or without calcium were studied. The calcium-binding capacity of phosphorylated soy peptide (SPP) reached 50.24 ± 0.20 mg/g. The result of computer stimulation and vibration spectrum showed that SPP could chelate with calcium by the phosphoric acid group, carboxyl oxygen of C-terminal Glu, Asp, and Arg, and phosphoric acid group of Ser on the SPP at a stoichiometric ratio of 1:1, resulting in the formation of the complex of ligand and peptide. Thermal stability showed that chelation enhanced peptide stability compared with SPP alone. Additionally, in vitro results showed that SPP-Ca could facilitate osteogenic proliferation and differentiation ability. SPP may function as a promising alternative to current therapeutic agents for bone loss.

Experiment and Simulation Reveal Residue Details for How Target Binding Tunes Calmodulin's Calcium-Binding Properties.

We aim to elucidate the molecular mechanism of the reciprocal relation of calmodulin's (CaM) target binding and its affinity for calcium ions (Ca2+), which is central to decoding CaM-dependent Ca2+ signaling in a cell. We employed stopped-flow experiments and coarse-grained molecular simulations that learn the coordination chemistry of Ca2+ in CaM from first-principle calculations. The associative memories as part of the coarse-grained force fields built on known protein structures further influence CaM's selection of its polymorphic target peptides in the simulations. We modeled the peptides from the Ca2+/CaM-binding domain of Ca2+/CaM-dependent kinase II (CaMKII), CaMKIIp (293-310) and selected distinctive mutations at the N-terminus. Our stopped-flow experiments have shown that the CaM's affinity for Ca2+ in the bound complex of Ca2+/CaM/CaMKIIp decreased significantly when Ca2+/CaM bound to the mutant peptide (296-AAA-298) compared to that bound to the wild-type peptide (296-RRK-298). The coarse-grained molecular simulations revealed that the 296-AAA-298 mutant peptide destabilized the structures of Ca2+-binding loops at the C-domain of CaM (c-CaM) due to both loss of electrostatic interactions and differences in polymorphic structures. We have leveraged a powerful coarse-grained approach to advance a residue-level understanding of the reciprocal relation in CaM, that could not be possibly achieved by other computational approaches.

Recombinant amelogenin peptide TRAP promoting remineralization of early enamel caries: An in vitro study.

Objective: To explore the regulatory effect of recombinant amelogenin peptide TRAP on the remineralization of early enamel carious lesions. Methods: Forty-eight bovine enamel blocks that prepared initial lesions in vitro were split at random into four groups for immersion treatment for 12days: 1) remineralizing medium; 2) studied peptide 1 (consisting of the N- and C-termini of porcine amelogenin) + remineralizing medium; 3) studied peptide 2 (TRAP) + remineralizing medium; 4) fluoride + remineralizing medium. After demineralization and remineralization immersion, each specimen's mean mineral loss and lesion depth were measured using micro-computed tomography (micro-CT). The changes in lesion depth (∆LD) and mineral gain (∆Z) were computed following remineralization. The enamel samples were then cut into sections and examined with polarized light microscopy (PLM). The cross-section morphology was observed by scanning electron microscopy (SEM). The crystal phase was analyzed by an X-ray micro-diffractometer (XRD). The calcium-binding properties were determined using isothermal titration calorimetry (ITC). Results: Micro-CT analysis revealed a significant reduction in mineral loss in the four groups following the remineralization treatment (p < 0.05). The treatment with fluoride resulted in the greatest ∆Z and ∆LD, whereas the treatment with a remineralizing medium showed the least ∆Z and ∆LD among all groups. The ∆Z and ∆LD of the studied peptide 1 and studied peptide 2 groups were greater than those of the remineralizing medium group. However, there was no significant difference between the studied peptide 1 and studied peptide 2 groups (p > 0.05). All of the samples that the PLM analyzed had a thickening of the surface layer. A negative birefringent band changed in the lesion's body. The SEM displayed that minerals were formed in all four groups of samples. The XRD results indicated that the products of remineralization of the studied peptide were hydroxyapatite crystals (HA). ITC showed that there were two binding modes between the calcium and peptide TRAP. Conclusion: This study confirmed the potential of the recombinant amelogenin peptide TRAP as a key functional motif of amelogenin protein for enamel remineralization and provided a promising biomaterial for remineralization in initial enamel carious lesion treatment.

Identification and functional analysis of three novel osteogenic peptides isolated from tilapia scale collagen hydrolysate

On the basis of our previous study that 133 peptides were identified from the tilapia scale peptide-calcium chelate, the potential osteogenic peptide monomers through the calcium-binding properties of peptides and molecular docking were screened, and the osteogenic activity and active mechanism of the peptides were further researched in this study. Three highly osteogenic peptides GPAGPHGPVG (844.4191 Da), APDPFRMY (995.4534 Da), and TPERYY (827.3813 Da) were screened. Molecular docking showed that the three osteogenic peptides had the same interaction sites in the epidermal growth factor receptor (EGFR), namely ARG 285, GLN 8, GLY 317, THR 406, and HIS 409. Compared to the blank control group, within 50 μg/mL of GPAGPHGPVG, APDPFRMY, and TPERYY increased the proliferation of MC3T3-E1 cells by changing the cell proportion in the S and G2/M phases, and the alkaline phosphatase (ALP) activity of MC3T3-E1 cells treated with 50 μg/mL of the three active peptides increased by 25%, 37%, and 56%, respectively. The three active peptides at 10 μg/mL concentration significantly promoted the mineralization of osteoblasts, and the mineralized calcium nodules increased by 166%, 161%, and 111%, respectively. TPERYY significantly increased the expression of osteogenic genes (osteocalcin (OCN), type I collagen (Col I α) and transcription factor (OSX)). Moreover, TPERYY significantly increased the mRNA and protein expression of β-catenin to 1.39 and 2.6 times of the blank control group, respectively, while decreased the mRNA and protein expression of glycogen synthesis kinase (GSK3β) to 1.6 and 2.3 times of the blank control group, respectively. This study provides a theoretical basis for using GPAGPHGPVG, APDPFRMY, and TPERYY peptides as functional foods to prevent osteoporosis.

CRELD2 is a novel modulator of calcium release and calcineurin-NFAT signalling during osteoclast differentiation

Cysteine rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER) resident chaperone protein with calcium binding properties. CRELD2 is an ER-stress regulated gene that has been implicated in the pathogenesis of skeletal dysplasias and has been shown to play an important role in the differentiation of chondrocytes and osteoblasts. Despite CRELD2 having an established role in skeletal development and bone formation, its role in osteoclasts is currently unknown. Here we show for the first time that CRELD2 plays a novel role in trafficking transforming growth factor beta 1 (TGF-β1), which is linked to an upregulation in the expression of Nfat2, the master regulator of osteoclast differentiation in early osteoclastogenesis. Despite this finding, we show that overexpressing CRELD2 impaired osteoclast differentiation due to a reduction in the activity of the calcium-dependant phosphatase, calcineurin. This in turn led to a subsequent block in the dephosphorylation of nuclear factor of activated T cells 1 (NFATc1), preventing its nuclear localisation and activation as a pro-osteoclastogenic transcription factor. Our exciting results show that the overexpression of Creld2 in osteoclasts impaired calcium release from the ER which is essential for activating calcineurin and promoting osteoclastogenesis. Therefore, our data proposes a novel inhibitory role for this calcium-binding ER-resident chaperone in modulating calcium flux during osteoclast differentiation which has important implications in our understanding of bone remodelling and the pathogenesis of skeletal diseases.

An acidic protein, Hf15, from Haliotis fulgens involved in biomineralization

Biomineralization leads to the hardening of mineralized materials, such as the shell of Mollusk, to fulfill a wide range of functions, such as (but not limited to) skeletal support, protection of the soft tissues, navigation, etc. The study of the proteins responsible for this process, shell matrix proteins (SMPs), allows addressing questions related to structure-function relationship and to the mechanism of mineral formation, which is limited in gastropod species. In this study, a low molecular weight protein was isolated from the insoluble fraction after decalcification with acetic acid of the shell of Haliotis fulgens and, named Hf15. The unglycosylated protein has a theoretical molecular weight of 15 kDa, it possesses calcium and chiting binding properties. Hf15 can precipitate calcium carbonate in vitro in presence of different salts. Analysis by LC-MS of the five peptide sequences of Hf15 generated by trypsinization revealed that two peptides displayed homology to an uncharacterized protein 3-like from Haliotis rufescens, Haliotis asinia and H. sorenseni. The results obtained indicated that Hf15 is a novel SMP involved in shell mineralization in Haliotis fulgens.

Alleviation of Surgery-Induced Osteitis in Sinonasal Cavity by Dexamethasone-Loaded Poly(lactic-co-glycolic acid) (PLGA) Microparticles with Strong Calcium-Binding Affinity.

For the treatment of sinus surgery-induced osteitis in chronic rhinosinusitis (CRS), oral or intranasal administration of corticoids is generally used, although it has critical limitations and unavoidable side effects. To overcome these limitations, we designed dexamethasone (Dex)-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles with bone-specific binding affinity, which could release the encapsulated Dex in a sustained manner on the exposed bone after the surgical wound in the nasal cavity. In a previous report, we prepared poly(butyl methacrylate-co-methacryloyloxyethyl phosphate) (PBMP) with both calcium-binding phosphomonoester groups and PLGA-binding butyl groups to introduce strong calcium-binding property to PLGA particles. In this study, after successful encapsulation of Dex in the PBMP-coated PLGA particles, we applied the Dex-PLGA/PBMP to the treatment of post-operative osteitis in the sinonasal cavity. The Dex-PLGA/PBMP showed more than 5-times higher binding affinity to the hydroxyapatite (HA) surface compared to the non-coated PLGA particles, without altering the morphology and encapsulation efficiency. After establishing the neo-osteogenesis mouse model by mechanical injury of the nasal mucosa, the activity of intranasally administered Dex-PLGA/PBMP was examined to inhibit the formation of undesirable new woven bone during the wound healing process. In addition, significantly lower osteocalcin activity was observed in the group treated with Dex-PLGA/PBMP, indicating decreased activation of osteoblasts. Overall, these results demonstrate that the PLGA/PBMP microparticle strategy has great potential for the treatment of CRS-related osteitis by localized corticoid delivery on the exposed bones with minimal side effects.

Evolution and Stress Responses of CLO Genes and Potential Function of the GhCLO06 Gene in Salt Resistance of Cotton.

The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT–PCR (qRT–PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT–PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.

Proteomic analysis of the secretome of equine herpesvirus-1 infected rabbit kidney cells

Herpesviruses are the main cause of abortions and respiratory or neurological disorders in horses. Various disease patterns are suspected to be associated with the A2254G point mutation in the DNA polymerase sequence (ORF30) of the herpesvirus genome, although the importance of this link is still under debate. Based on a label-free quantitative proteomic analysis, the differences in the secretion of some host proteins between rabbit kidney cells infected with A2254 and cells of the same line infected with G2254 equine herpesvirus 1 (EHV-1) strains were identified. In both groups, downregulation of proteins involved in insulin growth factor and extracellular matrix pathway regulation was observed. Among 12 proteins with increased secretion, 8 were regulated only in G2254 EHV-1 infection. Those were endoplasmic reticulum chaperones with calcium binding properties, related to unfolded protein response and mitochondria. It was presumed that the secretion of proteins such as calreticulin, Hspa5 or endoplasmin may contribute to the pathogenesis of EHV-1 infection.

Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from Toxoplasma gondii.

Centrins are calcium (Ca2+)-binding proteins that have been implicated in several regulatory functions. In the protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, three isoforms of centrin have been identified. While increasing information is now available that links the function of centrins with defined parasite biological processes, knowledge is still limited on the metal-binding and structural properties of these proteins. Herein, using biophysical and structural approaches, we explored the Ca2+ binding abilities and the subsequent effects of Ca2+ on the structure of a conserved (TgCEN1) and a more divergent (TgCEN2) centrin isoform from T. gondii. Our data showed that TgCEN1 and TgCEN2 possess diverse molecular features, suggesting that they play nonredundant roles in parasite physiology. TgCEN1 binds two Ca2+ ions with high/medium affinity, while TgCEN2 binds one Ca2+ with low affinity. TgCEN1 undergoes significant Ca2+-dependent conformational changes that expose hydrophobic patches, supporting a role as a Ca2+ sensor in toxoplasma. In contrast, Ca2+ binding has a subtle influence on conformational features of TgCEN2 without resulting in hydrophobic exposure, suggesting a different Ca2+ relay mode for this isoform. Furthermore, TgCEN1 displays a Ca2+-dependent ability to self-assemble, while TgCEN2 did not. We discuss our findings in the context of Ca2+ signaling in toxoplasma.

Arabinogalactan-proteins of Zostera marina L. contain unique glycan structures and provide insight into adaption processes to saline environments

Seagrasses evolved from monocotyledonous land plants that returned to the marine habitat. This transition was accomplished by substantial changes in cell wall composition, revealing habitat-driven adaption to the new environment. Whether arabinogalactan-proteins (AGPs), important signalling molecules of land plants, are present in seagrass cell walls is of evolutionary and plant development interest. AGPs of Zostera marina L. were isolated and structurally characterised by analytical and bioinformatics methods as well as by ELISA with different anti-AGP antibodies. Calcium-binding capacity of AGPs was studied by isothermal titration calorimetry (ITC) and microscopy. Bioinformatic searches of the Z. marina proteome identified 9 classical AGPs and a large number of chimeric AGPs. The glycan structures exhibit unique features, including a high degree of branching and an unusually high content of terminating 4-O-methyl-glucuronic acid (4-OMe GlcA) residues. Although the common backbone structure of land plant AGPs is conserved in Z. marina, the terminating residues are distinct with high amounts of uronic acids. These differences likely result from the glycan-active enzymes (glycosyltransferases and methyltransferases) and are essential for calcium-binding properties. The role of this polyanionic surface is discussed with regard to adaption to the marine environment.

Bioactivity Potentials and General Applications of Fish Protein Hydrolysates

Fish protein hydrolysates (FPHs) are rich source of amino acids and peptides that are beneficial to human health. Industrially, when fish are processed, huge amount of wastes are generated. These wastes are often discarded into waterways, seas, oceans or even buried in the ground. However, these wastes can be processed into FPHs using enzymatic hydrolysis. Generally, FPHs possess numerous bioactivity potentials such as antioxidant, antimicrobial, anti-tumor, ACE inhibiting activity, calcium binding properties, and anticoagulant properties. All of which were reviewed and presented in this article. In addition, their application in food, agriculture and pharmaceutical industries were explored. Nevertheless, despite the numerous bioactivities of FPHs, information on transportation and bioavailability of FPHs in gastro-intestinal system is limited. Thus, it was suggested that further work should be carried out in this aspect to validate their action, mechanism and therapeutic value in the gastro-intestinal system. This will enhance their potentials as a nutritive ingredient in the emerging nutraceutical and pharmaceutical commercial markets.

Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms.

SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis (Arabidopsis thaliana) AtSCS gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca2+ Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing 35S::AtSCS-A-c-myc or 35S::AtSCS-B-c-myc in the scs-1 knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins.

Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices.

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp-Glu-Ser), Nematoda (Asp-Asp-His) and Echinodermata (Asp-Glu-Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

Homology modeling and molecular dynamics simulations of a cassava translationally controlled tumor protein (MeTCTP)

Abiotic stresses are responsible for serious productivity and yield losses in many crops worldwide. Thus, there is great relevance in the prospection of plant molecular components, including gene products related to endogenous defense mechanisms, providing insights into plant tolerance. The TCTP (translationally controlled tumor protein) is a protein family highly conserved in eukaryotic organisms and related to abiotic stress responses. Recently, we have verified the thermotolerance in Escherichia coli and in vitro chaperone-like activity of TCTP from cassava (MeTCTP). To increase the understanding about the roles of MeTCTP in response to abiotic stresses, here we performed structural analysis of MeTCTP through homology modeling and molecular dynamics simulations, generating a three-dimensional model for calcium-binding to MeTCTP, since the calcium binding property is critical for the role of many proteins that are regulated in response to abiotic stress. Results presented here provided the first prediction for calcium-binding to MeTCTP by molecular dynamics simulations and the main residues related to this interaction. In addition, such results can contribute for the understanding of TCTP function against abiotic stress under in vivo conditions in cassava, as well as in other plants, such as Arabidopsis thaliana.

Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum.

Dantrolene interacts with ryanodine receptors in skeletal and cardiac muscle affecting sarcoplasmic reticulum calcium release. Since dantrolene is lipophilic it could also affect intra-sarcoplasmic reticulum calcium handling proteins such as calsequestrin. This study investigated whether dantrolene (1-30µmol/L) alters the polymerization state and the calcium binding capacity of recombinant cardiac calsequestrin and cardiac sarcoplasmic reticulum calcium handling. Human recombinant cardiac calsequestrin was used to make simultaneous measurements of turbidity (to indicate calsequestrin polymerization) and calcium binding to calsequestrin in the presence and absence of dantrolene. Caffeine-induced Ca2+ transients were used to investigate the effects of dantrolene on sarcoplasmic reticulum calcium loading and release in saponin-permeabilized cardiomyocytes laid down in monolayers in 96-well array plates. Dantrolene (1-30µmol/L) increased the polymerization state of calsequestrin and its calcium binding capacity. In the presence of dantrolene, calsequestrin-dependent turbidity increased 2.5-11.5-fold at 1.0mmol/L calcium added to unbuffered Ca2+ solutions and 3-10-fold when calcium was raised from 0.06 to 30µmol/L. The dantrolene-dependent increase in turbidity at 30µmol/L dantrolene was associated with a 3-fold increase in the number of calcium ions bound per calsequestrin molecule at 30 and 100µmol/L calcium. The caffeine-induced releasable calcium loaded by the sarcoplasmic reticulum at 0.63µmol/L free calcium in permeabilized cardiomyocytes was also increased in the presence of 30µmol/L dantrolene. Dantrolene alters the polymerization state and the calcium binding properties of cardiac calsequestrin and increases sarcoplasmic reticulum calcium loading in permeabilized cardiomyocytes.