Membrane binding of the neuronal calcium sensor recoverin – modulatory role of the charged carboxy-terminus

Background  

The Ca²⁺-binding protein recoverin operates as a Ca²⁺-sensor in vertebrate photoreceptor cells. It undergoes a so-called Ca²⁺-myristoyl switch when cytoplasmic Ca²⁺-concentrations fluctuate in the cell. Its covalently attached myristoyl-group is exposed at high Ca²⁺-concentrations and enables recoverin to associate with lipid bilayers and to inhibit its target rhodopsin kinase. At low Ca²⁺-concentrations the myristoyl group is inserted into a hydrophobic pocket of recoverin thereby relieving inhibitory constraint on rhodopsin kinase. Hydrophobic and electrostatic interactions of recoverin with membranes have not been clearly determined, in particular the function of the positively charged carboxy-terminus in recoverin ¹⁹¹QKVKEKLKEKKL²⁰² in this context is poorly understood.     

Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

Background  

Ca²⁺-ATPases of endoplasmic reticulum (SERCAs) are responsible for maintenance of the micro- to millimolar Ca²⁺ ion concentrations within the endoplasmic reticulum (ER) of eukaryotic cells. This intralumenal Ca²⁺ storage is important for the generation of Ca²⁺ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca²⁺ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity.     

Ca<Superscript>2+</Superscript> binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein

Background

The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca²⁺ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca²⁺ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca²⁺ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca²⁺ cage within CR-domains from this family of receptors that function in endocytosis and signalling.

Results

We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca²⁺ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca²⁺, and show that CR56 binds 2 Ca²⁺ ions with an average affinity of K_D = 10.6 microM, and there is no indication of additional Ca²⁺ binding sites within this receptor fragment.

Conclusions

Both CR-domains of CR56 bind a single Ca²⁺ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.

     

Release of Ca2+ and Mg2+ from yeast mitochondria is stimulated by increased ionic strength

Background  

Divalent cations are required for many essential functions of mitochondrial metabolism. Yet the transporters that mediate the flux of these molecules into and out of the mitochondrion remain largely unknown. Previous studies in yeast have led to the molecular identification of a component of the major mitochondrial electrophoretic Mg²⁺ uptake system in this organism as well as a functional mammalian homolog. Other yeast mitochondrial studies have led to the characterization of an equilibrative fatty acid-stimulated Ca²⁺ transport activity. To gain a deeper understanding of the regulation of mitochondrial divalent cation levels we further characterized the efflux of Ca²⁺ and Mg²⁺ from yeast mitochondria.     

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

Background

Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^st and 2^nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.

Results

The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵ and 5.0 × 10² M^-1 with ??Hs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^st non-canonical EF-hand is largely enhanced by the binding of Ca²⁺ to the 2^nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its ??-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.

Conclusions

We demonstrated that the 1^st non-canonical EF-hand of NOX5 has very weak Ca²⁺ binding affinity compared with the 2^nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺ specific.

Graphical abstract

     

Cluster analysis application identifies muscle characteristics of importance for beef tenderness

Background

Unsaturated fatty acids are susceptible to oxidation and damaged chains are removed from glycerophospholipids by phospholipase A₂. De-acylated lipids are then re-acylated by lysophospholipid acyltransferase enzymes such as LPCAT1 which catalyses the formation of phosphatidylcholine (PC) from lysoPC and long-chain acyl-CoA.

Results

Activity of LPCAT1 is inhibited by Ca²⁺, and a Ca²⁺-binding motif of the EF-hand type, EFh-1, was identified in the carboxyl-terminal domain of the protein. The residues Asp-392 and Glu-403 define the loop of the hairpin structure formed by EFh-1. Substitution of D³⁹² and E⁴⁰³ to alanine rendered an enzyme insensitive to Ca²⁺, which established that Ca²⁺ binding to that region negatively regulates the activity of the acyltransferase amino-terminal domain. Residue Cys-211 of the conserved motif III is not essential for catalysis and not sufficient for sensitivity to treatment by sulfhydryl-modifier agents. Among the several active cysteine-substitution mutants of LPCAT1 generated, we identified one to be resistant to treatment by sulfhydryl-alkylating and sulfhydryl-oxidizer agents.

Conclusion

Mutant forms of LPCAT1 that are not inhibited by Ca²⁺ and sulfhydryl-alkylating and ?Coxidizing agents will provide a better understanding of the physiological function of a mechanism that places the formation of PC, and the disposal of the bioactive species lysoPC, under the control of the redox status and Ca²⁺ concentration of the cell.     

1,2-Dichlorobenzene affects the formation of the phosphoenzyme stage during the catalytic cycle of the Ca<Superscript>2+</Superscript>-ATPase from sarcoplasmic reticulum

Background

1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca²⁺-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca²⁺-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca²⁺ concentration.

Results

Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca²⁺-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca²⁺ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca²⁺ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca²⁺ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-³²P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C.

Conclusions

The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E₂P?·?Ca₂ state to E₂ and P_i, which explains the uncoupling of the ATP hydrolysis from the Ca²⁺ transport.

     

The Metal Coordination of sCD39 during ATP Hydrolysis

Background  

The hydrolysis of ATP and ADP by ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) requires divalent cations, like Ca²⁺ and Mg²⁺. In spite of considerable work, it is not clear whether divalent cations bind to the enzyme in the absence of nucleotide or only as nucleotide-Me⁺² complex. Here we study the protein ligands for Me⁺².     

Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II

Background  

Intracellular free calcium ([Ca²⁺]_i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways.     

Beta-arrestin inhibits CAMKKbeta-dependent AMPK activation downstream of protease-activated-receptor-2

Background  

Proteinase-activated-receptor-2 (PAR₂) is a seven transmembrane receptor that can activate two separate signaling arms: one through Gαq and Ca²⁺ mobilization, and a second through recruitment of β-arrestin scaffolds. In some cases downstream targets of the Gαq/Ca²⁺ signaling arm are directly inhibited by β-arrestins, while in other cases the two pathways are synergistic; thus β-arrestins act as molecular switches capable of modifying the signal generated by the receptor.     

Luminescent core–shell Ca2MoO5:Eu3+-MCM-41 structure for sustained drug release

The spherical mesoporous MCM-41 coated with a novel Ca₂MoO₅:Eu³⁺ phosphor layer was prepared for the first time. The obtained Ca₂MoO₅:Eu³⁺-MCM-41 was characterized via XRD and FT-IR. The crystal system of the Ca₂MoO₅ phase was determined to be orthorhombic, and its space group was found to be Ima2 (46), and its cell parameters were a = 16.175, b = 5.1514, c = 5.6977 A°; α = β = γ = 90°. The particle dimensions of MCM-41 and Ca₂MoO₅:Eu³⁺-MCM-41 nanoparticles were determined to be 260 nm and 229 nm via scanning electron microscopy analysis. Bortezomib was loaded into the Ca₂MoO₅:Eu³⁺-MCM-41 nanoparticles under scCO₂ at 200 bars and 40 °C. The results of the TG analysis showed that the amount of drug-loaded to MCM-41 and Ca₂MoO₅:Eu³⁺-MCM-41 nanoparticles were determined to be 14.02% and 3.02%, respectively. The BET analysis showed that while the specific surface area and pore volume of MCM-41 and Ca₂MoO₅:Eu³⁺ before Bortezomib (BTZ) loading were 1,506 m²/g and 267 m²/g, respectively, after drug loading these values were found to decrease to 488 m²/g and 7.883 m²/g. It was determined that BTZ was released from the nanoparticles in a sustained manner over 66 h. The R² value, which was calculated to be 0.9739, indicated that the release kinetic of BTZ followed the Korsmeyer–Peppas model.     

High sensitivity and multicolor tunable optical thermometry in Bi3+/Eu3+ co-doped Ca2Sb2O7 phosphors

Currently, with increasing demand for non-contact fluorescence intensity ratio-based optical thermometry, a novel phosphor with high-efficiency, dual-emitting centers, and differentiable temperature sensitivity is more and more urgent to develop. In this work, an efficient dual-emitting center optical thermometry with high sensitivity and multicolor tunable in Ca₂Sb₂O₇:Bi³⁺, Eu³⁺ phosphor is firstly designed and successfully prepared. Under 330 nm excitation, the fabricated phosphor presents the featured and distinguishable emissions of Bi³⁺ and Eu³⁺ ions. The high efficiency energy transfer from Bi³⁺ to Eu³⁺ ions is proved and its corresponding mechanism belongs to dipole-dipole interaction. By modulating the ratio of Bi³⁺/Eu³⁺, the multicolor changes from blue to pink are realized. Based on the discriminative thermal quenching behavior between Bi³⁺ and Eu³⁺, the fluorescence intensity ratio of Eu³⁺ to Bi³⁺ in Ca₂Sb₂O₇ samples illustrates excellent optical thermometry performance from 298 to 523 K. The maximum absolute sensitivity (S_a) and relative sensitivity (S_r) reach as high as 0.2773 K^?1 at 523 K and 2.37% K^?1 at 448 K, respectively. Notably, the discriminated surrounding temperature can be directly confirmed by observing the emitting color from purple to orange-red with the temperature increase from 298 to 523 K. Furthermore, the as-prepared phosphor materials also demonstrate outstanding repeatability and excellent reversibility. These results exhibit that the designed Ca₂Sb₂O₇:Bi³⁺, Eu³⁺ phosphors have great promising applications in the field of non-contact optical temperature thermometry and thermochromic.     

Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50 and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca²⁺ and Cu²⁺ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca²⁺ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches. The K _m and V _max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis.     

Investigation of coagulation process of wet-spun sodium alginate polymannuronate fibers with varied functionality using organic coagulants and cross-linkers

Alginate fibers composed of mannuronate blocks were manufactured via a wet spinning process by varying organic coagulants and cross-linkers. As cross-linkers, both ionic (Ca²⁺, Ba²⁺, and Al³⁺) and covalent (citric acid) were used, and as coagulants, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and tetrahydrofuran (THF) were selected. Fiber properties depended on the ionic size, valence, and chemical structure of the cross-linkers. Among the used cross-linkers, Al³⁺ being the smallest ion, could diffuse inside the polymer solution easily and form metal-carboxylate coordination with three mannuronate chains. However, Ca²⁺ and Ba²⁺ both were very large compared to it, and polymer chains were positioned at about 48° for Ca²⁺ and in a parallel manner for Ba²⁺ after forming inter and intrachain metal-carboxylate bonds. Citric acid underwent an esterification reaction with the hydroxyl groups of mannuronate chains. Coagulants stabilized the cross-linking process and formed hydrogen bonds with the polymer chains. Depending on the cross-linkers and coagulants, the fiber diameter ranged from 217 μm to 830 μm and tensile modulus ranged from 88 MPa to 2 MPa. Ca²⁺, and then Ba²⁺ ions were more effective as cross-linkers since they produced fibers with superior mechanical properties followed by citric acid and Al³⁺. For all cross-linkers except citric acid, when DMSO was used as a coagulant, the tensile modulus was the highest. This indicates that DMSO better stabilized the cross-linking process during coagulation. In phosphate buffer saline (PBS), only Ba²⁺ cross-linked fibers could retain their original structure for 24 h, and fiber formed with coagulant DMSO swelled the least because of its compact structure. It also lost the least percentage of weight during 6 weeks. Thermal properties of the samples were also different as Ba²⁺ and Ca²⁺ cross-linked samples were more resistive to high temperature than other samples. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay illustrated the non-cytotoxicity of all the manufactured fibers.     

Purification and Biochemical Characterization of Polyphenol Oxidases from Embryogenic and Nonembryogenic Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) Cells

Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl₂, and FeCl₃ markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg²⁺, Ca²⁺, and Mn²⁺ and was moderately inhibited by Ba²⁺, Cu²⁺, and Zn²⁺. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.     

Synthesis and photoluminescence properties of tunable emission phosphors Ca4Si2O7F2:Ce3+

The Ce³⁺ activated phosphors Ca₄Si₂O₇F₂:Ce³⁺ are prepared by a solid state reaction technique. The UV–vis luminescence properties as well as fluorescence decay time spectra are investigated and discussed. The results revealed that there were two kinds of Ce³⁺ luminescence behavior with 408 and 470 nm emissions, respectively. Under 355 nm excitation, the Ce(1) emission (408 nm) is dominant at low doping concentration, and then the Ce(2) emission (470 nm) get more important with increasing of Ce³⁺ concentrations in the host. The phosphors Ca₄Si₂O₇F₂:xCe³⁺ show tunable emissions from blue area to green-blue area under near-ultraviolet light excitation, indicating a potential application in near-UV based w-LEDs.     

Engineering a subcellular targetable, red-emitting, and ratiometric fluorescent probe for Ca2+ and its bioimaging applications

A new, visible-light-excited and red-emitting fluorescent Ca²⁺ probe, STDBT, was synthesized, which consists of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid as a Ca²⁺-chelating moiety and two benzothiazolium hemicyanine dyes as fluorophores. The spectral profiles of its free and Ca²⁺-bound forms were studied. Upon addition of Ca²⁺, the fluorescence spectra of STDBT displayed a significant enhancement (about 48-fold) in fluorescence intensity and a 20-nm blueshift (from 600 to 580 nm) in the emission spectrum. Both the absorption and the excitation spectra of STDBT showed a very large (more than 100 nm) hypsochromic shift in the long-wavelength maxima upon binding with Ca²⁺. Interestingly, in contrast with the commonly used Ca²⁺ indicator Fluo-3, when the acetoxymethyl ester of STDBT enters into cells, it distributes both in the cytosol and the nucleus, but displays a very clear boundary between the two compartments. This allows STDBT to be used as a double targetable Ca²⁺ probe that can be used to report cytoplasmic Ca²⁺ and nuclear Ca²⁺ simultaneously.     

CHARACTERIZATION OF A CALCIUM-REQUIRING PHASE DURING PHYTOCHROME-MEDIATED FERN-SPORE GERMINATION OF Dryopteris paleacea Sw.*

Abstract— For phytochrome-mediated fern-spore germination in Dryopteris paleacea Sw., initiated by a saturating red light (R) irradiation 20 h after imbibition, an almost absolute requirement for extracellular Ca²+ is found. To investigate the kinetics of this Ca²+ requirement spores were sown on a Ca²+-free medium (Ca²+ < 10^?8M) and Ca²+ was raised to 1 mM at defined periods after R. Alternatively, spores were sown on a Ca²+-containing medium (1 mM) and transferred to a Ca²+-free medium. A clearly defined period for the Ca²+ requirement is found between 30 and 50 h after R, while Ca²+ had to be present in the medium for at least 9 h to obtain half-maximal germination. However, since the kinetics of deprivation and delayed addition of Ca²+ do not provide significantly different results, only a relative short presentation time for extracellular Ca²+ has to be expected at the level of a single cell. Ca²+ sensitivity is determined by the timing of the R irradiation, i.e. the timing of Pfr formation, which has been concluded from the observation that the variation of the imbibition time does not affect the kinetics of Ca²+ requirement and that the temporal application of polyethylene glycol (PEG), which is assumed to interrupt Pfr action, shifts the Ca²+ requirement to delayed intervals. These observations, as well as the fact that the requirement of Ca²+ has been observed for a limited period are interpreted as indirect evidence that Ca²+ action plays the role of a specific link in the phytochrome-mediated signal-transduction chain. The “Ca²+ kinetics” are compared with the kinetics of escape from reversibility by far-red (FR) light and with kinetics of basic cell physiological processes occurring during germination. “Escape kinetics”, indicating “coupling” of Pfr to subsequent dark reactions, are observed at significant earlier intervals, and a gap of about 15 h is found for both the action of Pfr and of Ca²+. Thus, the direct interaction of Pfr with external Ca²+ as a first transduction step can be excluded experimentally. The kinetics of chlorophyll formation are found to be only slightly delayed, whereas the kinetics of mitosis are shifted by approximately 30 h. Almost the same slope is obtained for all kinetics investigated so far and one reaction spans a period of approximately 35 h. Obviously, variability found in the spore population is due to the coupling of Pfr whereas subsequent reactions proceed with almost identical velocity.     

Synthesis of Ca<Subscript>3</Subscript>Al<Subscript>6</Subscript>Si<Subscript>2</Subscript>O<Subscript>16</Subscript>: Ce<Superscript>3+</Superscript>, Tb<Superscript>3+</Superscript> phosphors by sol–gel method and optical properties

Ca₃Al₆Si₂O₁₆: Ce³⁺, Tb³⁺ phosphors have been prepared by sol–gel method. The structure and photoluminescence properties were studied with careful. The results indicated that the single-phased Ca₃Al₆Si₂O₁₆ phosphors crystallize at 1,000 °C for 2 h in conventional furnace. With appropriate concentrations of Ce³⁺ and Tb³⁺ ions into Ca₃Al₆Si₂O₁₆ matrix, these materials exhibit blue phosphors and white light under ultraviolet radiation. White-light emission can be achieved because of a 400 nm emission ascribed to transitions of Ce³⁺ ions and three sharp peaks at 487, 543, 585 nm, respectively, resulting from transitions of Tb³⁺ ions.     

A highly selective fluorescence turn-on detection of Al3+ and Ca2+ based on a coumarin-modified rhodamine derivative

A fluorescent chemosensor 1 was synthesized containing a coumarin moiety bound to rhodamine B hydrazide. Compound 1 displayed different fluorescence emission responses to Al³⁺ and Ca²⁺ ions with high quantum yields (0.64 and 0.15, respectively) and low detection limits (3.0 × 10^–8 and 9.4 × 10^–8 M, respectively). The possible binding modes of compound 1 with Al³⁺ and Ca²⁺ ion were calculated using a Job plot, HRMS, ¹H NMR spectroscopic titration and IR spectroscopy. Moreover, the calcium in 1-Ca²⁺ could be displaced by Al³⁺ ions, resulting in another ratiometric sensing signal output, which indicates that 1-Ca²⁺ could detect Al³⁺ ions in a ratiometric way. Bioimaging results also demonstrated that compound 1 could act as an intracellular Al³⁺ ion imaging sensor.