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CorA is a Mg 2+ channel that plays a key role in the homeostasis of intracellular Mg 2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg 2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg 2+ , which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorA CD ). The Mg 2+ -binding sites of CorA CD differ depending on the species, suggesting that the Mg 2+ -binding modes and Mg 2+ -mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg 2+ -binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorA CD (cjCorA CD ). cjCorA CD adopts a three-layered α/β/α structure as observed in other CorA orthologs. Interestingly, cjCorA CD exhibited enhanced thermostability in the presence of Ca 2+ , Ni 2+ , Zn 2+ , or Mn 2+ in addition to Mg 2+ , indicating that cjCorA CD interacts with diverse divalent cations. This cjCorA CD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorA CD structure away from the pentamerization interface. Consistently, cjCorA CD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorA CD binds divalent cations in a unique pentamerization-independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Huntington's Disease (HD) is caused by an abnormal expansion of the polyglutamine (polyQ) domain within the first exon of the huntingtin protein (htt). This expansion promotes disease-related htt aggregation into amyloid fibrils and the formation of proteinaceous inclusion bodies within neurons. Fibril formation is a complex heterogenous process involving an array of aggregate species such as oligomers, protofibrils, and fibrils. In HD, structural abnormalities of membranes of several organelles develop. In particular, the accumulation of htt fibrils near the endoplasmic reticulum (ER) impinges upon the membrane, resulting in ER damage, altered dynamics, and leakage of Ca 2+ . Here, the aggregation of htt at a bilayer interface assembled from ER-derived liposomes was investigated, and fibril formation directly on these membranes was enhanced. Based on these observations, simplified model systems were used to investigate mechanisms associated with htt aggregation on ER membranes. As the ER-derived liposome fractions contained residual Ca 2+ , the role of divalent cations was also investigated. In the absence of lipids, divalent cations had minimal impact on htt structure and aggregation. However, the presence of Ca 2+ or Mg 2+ played a key role in promoting fibril formation on lipid membranes despite reduced htt insertion into and association with lipid interfaces, suggesting that the ability of divalent cations to promote fibril formation on membranes is mediated by induced changes to the lipid membrane physicochemical properties. With enhanced concentrations of intracellular calcium being a hallmark of HD, the ability of divalent cations to influence htt aggregation at lipid membranes may play a role in aggregation events that lead to organelle abnormalities associated with disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS]) expresses two homologous CDF efflux transporters, MntE and CzcD, which selectively transport Mn and Zn, respectively. We discovered that the MntE- and CzcD-deficient strains exhibited a marked decrease in the viability of macrophage-differentiated THP-1 cells and neutrophils. In addition, the viability of mice infected with both deficient strains markedly increased. Consistent with a previous study, our results suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular Mn levels and contributing to the growth of GAS. The maturation and proteolytic activity of streptococcal cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zinc and copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the CzcD-deficient strain. Furthermore, Mn inhibited SpeB maturation and proteolytic activity in a MntE-deficient strain. Since the host pathogenicity of the SpeB-deficient strain was significantly reduced, maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also play an essential role in its virulence. These findings provide new insights into the molecular mechanisms of pathogenicity, which allow pathogens to survive under stressful conditions associated with elevated metal ion concentrations during host infection., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Copper cations play fundamental roles in biological systems, such as protein folding and stabilization, or enzymatic reactions. Although copper is essential to the cell, it can become cytotoxic if present in too high concentration. Organisms have therefore developed specific regulation mechanisms towards copper. This is the case of the Pco system present in the bacterium Caulobacter crescentus, which is composed of two proteins: a soluble periplasmic protein PcoA and an outer membrane protein PcoB. PcoA oxidizes Cu + to Cu 2+ , whereas PcoB is thought to be an efflux pump for Cu 2+ . While the PcoA protein has already been studied, very little is known about the structure and function of PcoB. In the present work, PcoB has been overexpressed in high yield in E. coli strains and successfully refolded by the SDS-cosolvent method. Binding to divalent cations has also been studied using several spectroscopic techniques. In addition, a three-dimensional structure model of PcoB, experimentally supported by circular dichroism, has been constructed, showing a β-barrel conformation with a N-terminal disordered chain. This peculiar intrinsic disorder property has also been confirmed by various bioinformatic tools., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Protein arginine phosphorylation (pArg) is a relatively novel posttranslational modification. Protein arginine phosphatase YwlE negatively regulates arginine phosphorylation and consequently induces the expression of stress-response genes that are crucial for bacterial stress tolerance and pathogenic homolog Staphylococcus aureus virulence. However, little is known about the factors that affect the enzymatic activity of YwlE with the exception of the effect of oxidative stress. Herein, based on the hydrolysis of the chromogenic substrate p-nitrophenyl phosphate (pNPP) by YwlE, we investigate the role of metal cations and oxyanions in the regulation of YwlE activity. Interestingly, among the various cations that we tested, Ca 2+ activates YwlE, while other cations, including Ag + , Co 2+ , Cd 2+ , and Zn 2+ , are inhibitory. Furthermore, as chemical analogues of phosphate, oxyanions play multiple roles in phosphatase activity. The regulatory switch Cys within the catalytic site regulates YwlE activity. Specifically, the thiol of this Cys could be alkylated by IAM (iodoacetamide) or oxidized by H 2 O 2 , resulting in enzymatic inhibition. Conversely, reducing reagents, such as DTT (dithiothreitol), β-me (β-mercaptoethanol), and TCEP (tris(2-carboxyethyl)phosphine) enhance YwlE activity. Additionally, as a stable analogue to pArg, pAIE binds to YwlE with a K d of 149.1 nM and a binding stoichiometry n of 1.2 and inhibits YwlE with an IC 50 of 316.3 ± 12.73 μM. The inhibition and activation of YwlE may have broad implications for the physiology, pharmacology and toxicology of metal cations and oxyanions., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca 2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca 2+ channel that conducts L-type Ca 2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca 2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα 1S subunit abolishing Ca 2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn 2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca 2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca 2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca 2+ , Mn 2+ entry and subsequent quenching did occur because Mn 2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba 2+ and Ba 2+ currents were strongly blocked by external Ca 2+ . Ba 2+ permeation was smaller, current kinetics slower and Ca 2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca 2+ impeding in this way permeation. Because Ba 2+ binds with lower affinity to D, Ba 2+ currents occur, but with reduced amplitudes as compared to Ba 2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The SecA DEAD (Asp-Glu-Ala-Asp) motor protein uses binding and hydrolysis of adenosine triphosphate (ATP) to push secretory proteins across the plasma membrane of bacteria. The reaction coordinate of nucleotide exchange is unclear at the atomic level of detail., Methods: We performed multiple atomistic computations of the DEAD motor domain of SecA with different occupancies of the nucleotide and magnesium ion sites, for a total of ~1.7 μs simulation time. To characterize dynamics at the active site we analyzed hydrogen-bond networks., Results: ATP and ADP can bind spontaneously at the interface between the nucleotide binding domains, albeit at an intermediate binding site distinct from the native site. Binding of the nucleotide is facilitated by the presence of a magnesium ion close to the glutamic group of the conserved DEAD motif. In the absence of the magnesium ion, protein interactions of the ADP molecule are perturbed., Conclusions: A protein hydrogen-bond network whose dynamics couples to the occupancy of the magnesium ion site helps guide the nucleotide along the nucleotide exchange path. In SecA, release of magnesium might be required to destabilize the ADP binding site prior to release of the nucleotide., General Significance: We identified dynamic hydrogen-bond networks that help control nucleotide exchange in SecA, and stabilize ADP at an intermediate site that could explain slow release. The reaction coordinate of the protein motor involves complex rearrangements of a hydrogen-bond network at the active site, with perturbation of the magnesium ion site likely occurring prior to the release of ADP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although cultured adult cardiac myocytes in combination with cell-level genetic modifications have been adopted for the study of protein function, the cellular alterations caused by the culture conditions themselves need to be clarified before we can interpret the effects of genetically altered proteins. We systematically compared the cellular morphology, global Ca 2+ signaling, elementary Ca 2+ release (sparks), and arrangement of ryanodine receptor (RyR) clusters in short-term (2 days)-cultured adult rat ventricular myocytes with those of freshly isolated myocytes. The transverse (t)-tubules were remarkably decreased (to ∼25%) by culture, and whole-cell capacitance was reduced by ∼35%. The magnitude of depolarization-induced Ca 2+ transients decreased to ∼50%, and Ca 2+ transient decay was slowed by culture. The culture did not affect sarcoplasmic reticulum (SR) Ca 2+ loading. Therefore, fractional Ca 2+ release was attenuated by culture. In the cultured cells, the L-type Ca 2+ current (I Ca ) was smaller (∼50% of controls) and its inactivation was slower. In cultured myocytes, there were significantly fewer (∼50% of control) Ca 2+ sparks, the local Ca 2+ releases through RyR clusters, compared with in freshly isolated cells. Amplitude and kinetics (duration and time-to-peak) of individual sparks were similar, but they showed greater width in cultured cells. Immunolocalization analysis revealed that the cross-striation of RyRs distribution became weaker and less organized, and that the density of RyR clusters decreased in cultured myocytes. Our data suggest that the loss of t-tubules and generation of compromised Ca 2+ transients and I Ca in short-term adult ventricular cell culture are independent of SR Ca 2+ loading status. In addition, the deteriorated arrangement of the RyR-clusters and their decreased density after short-term culture may be partly responsible for fewer Ca 2+ sparks and a decrease in global Ca 2+ release., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca 2+ homeostasis. This work examines the kinetic parameters of Ca 2+ transport and the opening of the Ca 2+ -dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca 2+ uniport, with the kinetics of Na + -dependent Ca 2+ efflux not changing. The data obtained indicate that the decreased rate of Ca 2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca 2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca 2+ homeostasis of skeletal-muscle mitochondria., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca 2+ ) influx and spontaneous Ca 2+ oscillations. The oscillations cease during maturation but Ca 2+ influx continues, as the oocytes' internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca 2+ influx has not been completely determined. GV and matured oocytes are known to express three Ca 2+ channels, Ca V 3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca 2+ homeostasis, suggesting a complex regulation of Ca 2+ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca V 3.2 and TRPM7 channels contributed the majority of Ca 2+ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca 2+ entry. Sr 2+ influx was promoted by Ca V 3.2 channels, as Sr 2+ oscillations were negligible in Ca V 3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn 2+ entry relied on expression of Ca V 3.2 and TRPM7 channels, but Ni 2+ entry depended on the latter. Ca V 3.2 and TRPV3 channels combined to fill the Ca 2+ stores, although Ca V 3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca 2+ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca 2+ concentration, therefore, the use of Ca 2+ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca 2+ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS). Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca 2+ -triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest concerning this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Identifying and detecting similar target cations through combining "turn on" and "turn off" fluorescence mechanism is effective and challenging. Now a new colorimetric and ON-OFF-ON fluorescent probe N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-3-hydroxy-2-naphthohydrazide (L) was reported, which could detect Cu 2+ and Co 2+ in phosphate buffered CH 3 CH 2 OH-H 2 O solvent system. With the assistance of glutathione and pH adjustment, a unique ON-OFF-ON fluorescence detection strategy could be achieved for distinguishing Cu 2+ and Co 2+ . The emission of probe could recover from the L-Cu 2+ and L-Co 2+ system by addition of GSH or adjusting pH value to 4, respectively, which is due to the abolishment of paramagnetic Cu 2+ /Co 2+ . Based on fluorescence titration experiments, the limit of detection was determined as 3.84 × 10 -9  M and 4.55 × 10 -9  M for Cu 2+ and Co 2+ , respectively. Meanwhile, the detection limit reached 6.21 × 10 -8  M for Cu 2+ and 6.96 × 10 -8  M for Co 2+ according to absorbance signal output. Fast recognition of Cu 2+ /Co 2+ can be achieved by obvious color changes from green to colorless under UV light, as well as from yellow to orange-red in room light. The binding mode of L toward Cu 2+ and Co 2+ have been systematically studied by Job's plot analysis, ESI-MS, IR and density functional theory calculations. Most strikingly, further practical applications of the probe L in fluorescence imaging were investigated in MCF-7 cells and zebrafish due to its low cytotoxicity and good optical properties, suggesting that L could serve as a fluorescent sensor for tracking Cu 2+ and Co 2+ in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Two novel coumarin based fluorescent sensors CHP and CHS have been synthesized for the sequential detection of Zn 2+ ion and phosphate anion (PA) in DMF/HEPES buffer medium (1/5 v/v, 10 mM, pH = 7.4). On the addition of Zn 2+ ion to the solution of CHP or CHS resulted in a pronounced fluorescence enhancement, accompanying noticeable color change (under UV or daylight), while there was hardly obvious change with other competing metal ions co-existing. The detection limits (DL) of CHP and CHS toward Zn 2+ were separately determined as 1.03 × 10 -7 (R 2  = 0.9886) and 1.87 × 10 -7 (R 2  = 0.9902). The PET binding processes were affirmed by spectroscopic techniques, HRMS experiments and theoretical calculations. Subsequently, the CHP-Zn 2+ or CHS-Zn 2+ complexes showed high selectivity fluorescence quenching toward PA by snatching Zn 2+ ion from its complex and the binding processes were reversible. DLs were calculated as 2.07 × 10 -7  M (R 2  = 0.9928) and 2.63 × 10 -7  M (R 2  = 0.9954), respectively. Furthermore, the cell imaging experiments demonstrated that the sensors were capable of detecting of Zn 2+ and PA in vitro cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The pH-Low Insertion Peptide (pHLIP) has emerged as an important tool for targeting cancer cells; it has been assumed that its targeting mechanism depends solely on the mild acidic environment surrounding tumors. Here, we examine the role of Ca 2+ and Mg 2+ on pHLIP's insertion, cellular targeting, and drug delivery. We demonstrate that physiologically relevant concentrations of either cation can shift the protonation-dependent transition by up to several pH units toward basic pH and induce substantial protonation-independent transmembrane insertion of pHLIP at pH as high as 10. Consistent with these results, the ability of pHLIP to deliver the cytotoxic compound monomethyl-auristatin-F to HeLa cells is increased several fold in presence of Ca 2+ . Complementary measurements with model membranes confirmed this Ca 2+ /Mg 2+ -dependent membrane-insertion mechanism. The magnitude of this alternative Ca 2+ /Mg 2+ -dependent effect is also modulated by lipid composition-specifically by the presence of phosphatidylserine-providing new clues to pHLIP's unique tumor-targeting ability in vivo. These results exemplify the complex coupling between protonation of anionic residues and lipid-selective targeting by divalent cations, which is relevant to the general signaling on membrane interfaces., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A 2 (cPLA 2 ɛ) was found to be Ca 2+ -dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA 2 ɛ, and used these cells to analyze endogenous substrates and products of cPLA 2 ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca 2+ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA 2 ɛ Ca 2+ -dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca 2+ -uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca 2+ -uptake function. Thus, it is essential for the Ca 2+ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca 2+ -uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel carbohydrate binding module (CBM) was identified in a β-1,3-xylanase from Flammeovirga pacifica, which showed only 25.0% sequence identity with the reported CBMs with the coverage of 36.4%. To verify its function, a truncated β-1,3-xylanase (Xy13088-T) and a carbohydrate binding module (CBM3088) were expressed and purified. The thermostability and catalytic efficiency of the Xy13088-T declined significantly when compared with the full-length one, with the decreasing of the half-life and catalytic efficiency (Kcat/K m ) by 90%. Interestingly, the CBM3088 showed the binding ability to β-1,3-xylan only when Ca 2+ existed, which was different from the reported CBMs of β-1,3-xylanases. The maximum amount of CBM3088 binding to β-1,3-xylan was 9.65 μmol/g of β-1,3-xylan. The residues probably involved in the binding to the β-1,3-xylan and Ca 2+ were addressed by bioinformatics analysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oxidative aggregation of γ-crystallins induced by copper in aged lens increases the lens opacity and causes cataract formation. Therefore, chelation of free Cu 2+ by small molecules can inhibit metal-mediated aggregation of γ-crystallin. In this work, the inhibition potency of several naturally occurring flavonoid compounds was studied against aggregation of human γD-crystallin (HGD) mediated by copper ions. Among them, rutin demonstrated ~20% inhibition of HGD aggregation induced by Cu 2+ through its metal chelation ability. Not only that, the chaperone activity of lens chaperone, human αA-crystallin (HAA) was found to be enhanced in the presence of rutin. Subsequently, the molecular interactions between HAA and rutin were investigated using fluorescence and CD spectroscopy to understand the molecular basis of the chaperone activity enhancement by rutin. Quenching of HAA fluorescence by rutin with a quenching constant in the order of ~10 5  M -1 depicts a complexation between them. Entropy driven process of complexation between HAA and rutin suggests significant involvement of hydrophobic interactions. Fluorescence resonance energy transfer between protein and ligand can occur at a distance of 2.73 nm. Synchronous fluorescence and circular dichroism spectroscopy revealed that protein-ligand interaction does not cause any notable conformational changes in HAA. Experimental observations have been well substantiated by docking., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Accumulation of cadmium ions may result in adverse effects on plant due to the oxidative stress via destructions of antioxidants and antioxidant enzymes. As the core component of the glutathione antioxidant system, glutathione S-transferases (GSTs) have been reported as biomarkers for evaluating the metal-induced oxidative damage to plants, but the potential toxicity and underlying toxic molecular mechanisms remain unknown. This article investigated the molecular interactions of cadmium ions with Arabidopsis thaliana glutathione S-transferase phi8 (AtGSTF8) by multi-spectroscopic techniques and enzyme activity measurements. The intrinsic fluorescence of AtGSTF8 was quenched statically upon the addition of cadmium ions accompanied with the complex formation and structural and conformational alterations from multiple spectroscopic measurements, resulting in deconstructed protein skeleton and microenvironmental alterations around the Tyr and Trp residues. A single binding site was predicted for AtGSTF8 towards cadmium ions and the van der Walls interactions and hydrogen bonds are the major driving forces of the interaction. In addition, the transferase activity changes of AtGSTF8 upon the addition of cadmium ions have been observed. The implementation of this work helps to clarify the mechanism of oxidative damage and antioxidant enzymes response induced by heavy metal accumulation in plant at molecular level., (Copyright © 2019. Published by Elsevier B.V.)

UVB-induced skin damage is attributable to reactive oxygen species, which are triggered by intracellular Ca 2+ signals. However, exactly how the reactive oxygen species are triggered by intracellular Ca 2+ upon UVB irradiation remains obscure. Here, we show that UVB induces Ca 2+ signals via sequential generation of the following Ca 2+ messengers: inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose. UVB induced H 2 O 2 production through NADPH oxidase 4 activation, which is downstream to inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate. H 2 O 2 derived from NADPH oxidase 4 activated CD38 to produce cyclic ADP-ribose. UVB first evoked the pannexin channel to release ATP, which acts on P2X7 receptor to generate inositol 1,4,5-trisphosphate. Inhibitors of these messengers, as well as antioxidants, blocked UVB-induced Ca 2+ signals and IL-1β secretion in keratinocytes. Furthermore, ablation of CD38 and NADPH oxidase 4 protected against UVB-induced inflammation and IL-1β secretion in the murine epidermis. These results show that UVB induces IL-1β secretion through cross-talk between Ca 2+ and reactive oxygen species, providing insight towards potential targets against UVB-induced inflammation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Ca 2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca 2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca 2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca 2+ -binding protein which was called the Ca 2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca 2+ , which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have constructed a new coumarin based fluorescence probe BENZPYR with ICT character through condensation of N, N-diethylamino-3-acetyl coumarin with 2-hydrazinobenzothiazole. The absorbance and fluorescence spectral characteristics of BENZPYR revealed that the chemosensor can specifically detect for Cu 2+ ions over other different metal ions and the lowest limit of detection was found in nano molar range. The turn off sensor of BENZPYR is related to chelation enhanced quenching (CHEQ) and intramolecular charge transfer (ICT) processes were serve as excellent fluorescent detection of Cu 2+ ions in DMF medium. Fluorescence microscopy experiments revealed that probe BENZPYR may have application as a fluorophore to detect the Cu 2+ in living cells. The simulated DFT analysis of electronic and structural properties and also UV-vis absorption spectra are in well accordance with the experimental UV-vis absorption spectra., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Copper is an important element indispensable for human life and health. Many copper-determining probes have been created for exploring its functional behavior in various cell types but few of them contains both fluorescent and colorimetric characters. In the present study, we developed a set of copper probes by synthesizing several novel thiophene-based Schiff bases in order to make a suitable sensor for quantifying and imaging copper in living cells. We find that the ligand FS-1 has a splendid selectivity and affinity toward Cu 2+ among the common divalent metal ions. Living cell imaging show that FS-1 has a robust and repetitive fluorescence response in the presence of Cu 2+ only in the cytosolic space of Hepg2 cell and not in the other cells examined. These data suggest that we have developed a new copper probe that can be used as a Cu 2+ fluorescent and colorimetric sensor for in vivo and in vitro copper studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protein phosphatase 5 (PP5), an important member of serine/threonine protein phosphatases, has been associated with diverse human cancers. Structural data demonstrate that two metal ions and two water molecules are located in the active site of PP5 and are involved in the dephosphorylations of the substrate. Recently, the catalytic reaction of PP5 has been studied by a density functional theory (DFT) method, and the role of the water molecules in regulating the substrate binding of PP5 was explored by our previous molecular dynamics (MD) study. These two studies enrich our understanding of the catalytic mechanism of PP5. However, the questions of how the metal ions regulate the substrate binding and the contribution of each metal ion to the catalysis remain not answered. To this end, the role of the catalytic Mn 2+ ions in regulating the binding of PP5 with its substrate was investigated through MD simulations. Results reveal that the different states of Mn 2+ ions can alter the structure of the active site and the conformations of the residues coordinating the Mn 2+ ions or the substrate. This leads to changes in the secondary structure and dynamical fluctuation of PP5, especially for that of helix αJ. With the position change of helix αJ, the volume of the substrate binding pocket is altered by the different states of Mn 2+ ions, indicating the regulatory role of Mn 2+ ions in PP5 activity. Furthermore, combined with the simulation data of our previous study, the sequential steps for PP5 binding of the substrate, the two active site water molecules, and the two Mn 2+ ions are proposed. This may help to develop new inhibitors to treat the PP5-related cancers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A broad range of biochemicals, from proteins to nucleic acids, function properly only when associated with a metal, usually a divalent cation. Not any divalent metal will do: these metals differ in their ionic radius, dissociation in water, ionization potential, and number of unpaired electrons in their outer shells, and so substituting one metal for another often changes substrate positioning, redox reactivities, and physiological performance, and thus may serve as a regulatory mechanism. For instance, exchanging manganese for magnesium in several chloroplast enzymes maintains plant carbon-nitrogen balance under rising atmospheric CO 2 concentrations. Here, we review this and a few other cases where association of proteins or nucleic acids with different metals control metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg 2+ , the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn 2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mycobacterium leprae uptakes various bivalent metal ions via different transporters in host species. Uptake of Cu 2+ and Zn 2+ are essential for generation of superoxide dismutases and catalases, which provide defense against reactive oxygen species mediated death of this pathogen in macrophages. Furthermore, it has also been noticed that levels of different bivalent metal ions (Ca 2+ , Mg 2+ , Cu 2+ and Zn 2+ ) in blood serum are altered in leprotic patients. Mycobacterium leprae HSP18 is an immunodominant antigen which helps in growth and survival of Mycobacterium leprae in host species. A possible link can exist between HSP18 and aberration of bivalent metal ion homeostasis. Therefore, we investigated the interaction of these four bivalent metal ions with HSP18 and found that the protein only interacts with Zn 2+ and Cu 2+ . Such association process is reversible and moderately high affinity in nature with unit binding stoichiometry. Theoretical studies revealed that the most probable site for Zn 2+ -binding lies in the N-terminal domain; While, the same for Cu 2+ -binding lies in the "α-crystallin domain" of HSP18. Binding of Zn 2+ /Cu 2+ to HSP18 brings about subtle changes in the secondary and tertiary structure of HSP18 but are distinctly opposite in nature. While Zn 2+ causes oligomeric association, Cu 2+ leads to oligomeric dissociation of HSP18. Structural stability, surface hydrophobicity and chaperone activity of HSP18 are enhanced on Zn 2+ binding, while all of them are reduced upon Cu 2+ binding. Altogether, metal ions binding to HSP18 regulate its function which may have far reaching effect on the survival and pathogenicity of Mycobacterium leprae in host species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K + channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transient receptor potential (TRP) ion channels were first characterized on neurons, where they are classically implicated in sensory functions; however, research in recent decades has shown that many of these channels are also expressed on nonneuronal cell types. Emerging findings have highlighted the role of TRP channels in the skin, where they have been shown to be important in numerous cutaneous functions. Of particular interest is TRPV3, which was first described on keratinocytes. Its functional importance was supported when its gain-of-function mutation was linked to Olmsted syndrome, which is characterized by palmoplantar keratoderma, periorifacial hyperkeratosis, diffuse hypotrichosis and alopecia, and itch. Despite these exciting results, we have no information about the role and functionality of TRPV3 on keratinocytes at the cellular level. In this study, we identified TRPV3 expression both on human skin and cultured epidermal keratinocytes. TRPV3 stimulation was found to function as a Ca 2+ -permeable ion channel that suppresses proliferation of epidermal keratinocytes and induces cell death. Stimulation of the channel also triggers a strong proinflammatory response via the NF-κB pathway. Collectively, our data show that TRPV3 is functionally expressed on human epidermal keratinocytes and that it plays a role in cutaneous inflammatory processes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Divalent metals play important roles in maintaining metabolism and cellular growth of both eukaryotic hosts and invading microbes. Both metal deficiency and overload can result in abnormal cellular function or damage. Given its central role in host-pathogen interactions, subtle alterations of divalent metal homeostasis can occur in the course of infectious diseases which aim, from the host perspective, either to reduce the availability of respective metals to microbes or to use toxic metal accumulation to eliminate pathogens., Aims: To provide the reader with background information and clinical data on divalent metal homeostasis in host-pathogen interactions, how this affects the course of infectious disease and whether correction of metal disturbances has shown benefit in infections., Sources: An in-depth analysis of PubMed articles related to the topic of this review published in English between 1970 and 2016 was performed., Content: From the microbial perspective, divalent metals are essential for growth and pathogenicity and to mount effective protection against antimicrobial host responses, including toxic radical formation. Microbes have evolved multiple strategies to control their access to divalent metals. From the clinical perspective, alterations of divalent metal levels may result in increased or decreased susceptibility to infection and often occur in response to infections. However, keeping in mind the strategies underlying such alterations, for which the term 'nutritional immunity' was coined, the uncritical correction of such divalent metal imbalances may cause harm to patients. This review addresses the role of the divalent metals iron, selenium, zinc, manganese and copper in infectious diseases from a mechanistic and clinical perspective., Implications: We point out areas of research needed to expand our limited knowledge, hoping to improve the clinical management of patients with infections and to identify promising new targets for treatment by modulation of host or microbe divalent metal metabolism., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg 2+ /ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg 2+ /ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A pathological hallmark of Alzheimer disease and other tauopathies is the formation of neurofibrillary tangles mainly composed of bundles of fibrils formed by microtubule-associated protein Tau. Here we study the effects of Zn 2+ on abnormal aggregation and cytotoxicity of a pathological mutant ΔK280 of full-length human Tau. As revealed by Congo red binding assays, transmission electron microscopy, immunofluorescence, Western blot, and immunogold electron microscopy, pathological concentration of Zn 2+ dramatically accelerates the fibrillization of ΔK280 both in vitro and in SH-SY5Y neuroblastoma cells. As evidenced by annexin V-FITC apoptosis detection assay and MTT reduction assay, pathological concentration of Zn 2+ remarkably enhances ΔK280 fibrillization-induced apoptosis and toxicity in SH-SY5Y cells. Substitution of Cys-291 and Cys-322 with Ala, however, essentially eliminates such enhancing effects of Zn 2+ on the fibrillization and the consequent cytotoxicity of ΔK280. Furthermore, Zn 2+ is co-localized with and highly enriched in amyloid fibrils formed by ΔK280 in SH-SY5Y cells. The results from isothermal titration calorimetry show that Zn 2+ binds to full-length human Tau by interacting with Cys-291 and Cys-322, forming a 1:1 Zn 2+ -Tau complex. Our data demonstrate that zinc dramatically accelerates abnormal aggregation of human Tau and significantly increases Tau toxicity in neuronal cells mainly via bridging Cys-291 and Cys-322. Our findings could explain how pathological zinc regulates Tau aggregation and toxicity associated with Alzheimer disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The anionic-polyelectrolyte nature of the wall of Gram-positive bacteria has long been suspected to be involved in homeostasis of essential cations and bacterial growth. A better understanding of the coupling between the biophysics and the biology of the wall is essential to understand some key features at play in ion-homeostasis in this living system., Methods: We consider the wall as a polyelectrolyte gel and balance the long-range electrostatic repulsion within this structure against the penalty entropy required to condense cations around wall polyelectrolytes. The resulting equations define how cations interact physically with the wall and the characteristic time required for a cation to leave the wall and enter into the bacterium to enable its usage for bacterial metabolism and growth., Results: The model was challenged against experimental data regarding growth of Gram-positive bacteria in the presence of varying concentration of divalent ions. The model explains qualitatively and quantitatively how divalent cations interact with the wall as well as how the biophysical properties of the wall impact on bacterial growth (in particular the initiation of bacterial growth)., Conclusion: The interplay between polymer biophysics and the biology of Gram positive bacteria is defined for the first time as a new set of variables that contribute to the kinetics of bacterial growth., General Significance: Providing an understanding of how bacteria capture essential metal cations in way that does not follow usual binding laws has implications when considering the control of such organisms and their ability to survive and grow in extreme environments., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn 2+ -dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn 2+ , rather than Mg 2+ , as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn 2+ and Mg 2+ , respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn 2+ than with Mg 2+ . Notably, the substitution fidelity of PrimPol in the presence of Mn 2+ was determined to be in the range of 3.4×10 -2 to 3.8×10 -1 , indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn 2+ and 150-4500 with Mg 2+ , and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The tertiary structure of the GTPase center (GAC) of 23S ribosomal RNA (rRNA) as seen in cocrystals is extremely compact. It is stabilized by long-range hydrogen bonds and nucleobase stacking and by a triloop that forms within its three-way junction. Its folding pathway from secondary structure to tertiary structure has not been previously observed, but it was shown to require Mg 2+ ions in equilibrium experiments. The fluorescent nucleotide 2-aminopurine was substituted at selected sites within the 60-nt GAC. Fluorescence intensity changes upon addition of MgCl 2 were monitored over a time-course from 1ms to 100s as the RNA folds. The folding pathway is revealed here to be hierarchical through several intermediates. Observation of the nucleobases during folding provides a new perspective on the process and the pathway, revealing the dynamics of nucleobase conformational exchange during the folding transitions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In typical nonlinear plasma protein binding (PPB) behavior, the free fraction increases with increasing total concentrations. In contrast, when a drug exhibits atypical nonlinear PPB behavior, the free fraction decreases with increasing total concentrations. Tigecycline, a novel glycylcycline, exhibits atypical nonlinear PPB behavior, but the mechanism of such behavior is currently unknown. Because tigecycline can form complexes with metal ions, an interaction between metal ion, tigecycline, and plasma proteins was hypothesized but not further investigated. The current work explores the role of metal ions in the atypical nonlinear PPB behavior of tigecycline and proposes a plausible mechanism of atypical nonlinear PPB behavior. The addition of ethylenediaminetetraacetic acid resulted in 10- to 30-fold higher unbound fractions, and the atypical behavior was nullified. The saturation of ethylenediaminetetraacetic acid chelation, by addition of excessive divalent metal ions, such as calcium and magnesium, led to the return of the atypical nonlinear PPB behavior. Different possible mechanisms were evaluated by simulation, and a plausible mechanism was proposed., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The interaction of natural lipopeptide pseudofactin II with a series of doubly charged metal cations was examined by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) mass spectrometry and molecular modelling. The molecular modelling for metal-pseudofactin II provides information on the metal-peptide binding sites. Overall, Mg(2+), Ca(2+) and Zn(2+) favor the association with oxygen atoms spanning the peptide backbone, whereas Cu(2+) is coordinated by three nitrogens. Circular dichroism (CD) results confirmed that Zn(2+) and Cu(2+) can disrupt the secondary structure of pseudofactin II at high concentrations, while Ca(2+) and Mg(2+) did not essentially affect the structure of the lipopeptide. Interestingly, our results showed that the addition of Zn(2+) and Cu(2+) helped smaller micelles to form larger micellar aggregates. Since pseudofactin II binds metals, we tested whether this phenomena was somehow related to its antimicrobial activity against Staphylococcus epidermidis and Proteus mirabilis. We found that the antimicrobial effect of pseudofactin II was increased by supplementation of culture media with all tested divalent metal ions. Finally, by using Gram-positive and Gram-negative bacteria we showed that the higher antimicrobial activity of metal complexes of pseudofactin II is attributed to the disruption of the cytoplasmic membrane., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23IMS) and the interaction of presequence peptides with Tim23IMS in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23IMS. The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim23IMS is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca(2+) is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M(2+): Mg(2+), Ca(2+), Sr(2+) or Ba(2+)). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M(2+). Moreover, we performed competition experiments by varying the concentration of luminal M(2+) (Mg(2+), Sr(2+) or Ba(2+)) from 8 mM to 53 mM and investigated its ability to compete with 1mM luminal Ca(2+). We demonstrate that all tested M(2+) bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca(2+)>Sr(2+)>Mg(2+)~Ba(2+), showing a strong correlation with the M(2+) affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gallic acid, a polyhydroxylphenolic compound, is widely distributed in various plants, fruits and foods. It has been shown that gallic acid passes into blood brain barrier and reaches the brain tissue of middle cerebral artery occlusion rats. However, the effect of gallic acid on Ca(2+) signaling in glia cells is unknown. This study explored whether gallic acid affected Ca(2+) homeostasis and induced Ca(2+)-associated cytotoxicity in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. Gallic acid (20-40 μM) concentration-dependently induced cytotoxicity and intracellular Ca(2+) level ([Ca(2+)]i) increases in DBTRG-05MG cells but not in CTX TNA2 cells. In DBTRG-05MG cells, the Ca(2+) response was decreased by half by removal of extracellular Ca(2+). In Ca(2+)-containing medium, gallic acid-induced Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (2-APB, econazole and SKF96365). In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished gallic acid-induced [Ca(2+)]i increases. Conversely, incubation with gallic acid also abolished thapsigargin-induced [Ca(2+)]i increases. Inhibition of phospholipase C with U73122 abolished gallic acid-induced [Ca(2+)]i increases. Gallic acid significantly caused cytotoxicity in DBTRG-05MG cells, which was partially prevented by prechelating cytosolic Ca(2+) with BAPTA-AM. Moreover, gallic acid activated mitochondrial apoptotic pathways that involved ROS production. Together, in DBTRG-05MG cells but not in CTX TNA2 cells, gallic acid induced [Ca(2+)]i increases by causing Ca(2+) entry via 2-APB, econazole and SKF96365-sensitive store-operated Ca(2+) entry, and phospholipase C-dependent release from the endoplasmic reticulum. This Ca(2+) signal subsequently evoked mitochondrial pathways of apoptosis that involved ROS production., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5' end of precursor tRNAs in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2 and PRORP3) in both its nucleus and its organelles; PRORP2 and PRORP3 localize to the nucleus and PRORP1 localizes to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here, we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2Å) revealing an overall V-shaped protein and conserved metallonuclease active-site structure. Our biochemical studies indicate that PRORP2 requires Mg(2+) for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single-turnover conditions. We also demonstrate that PRORP2 preferentially binds precursor tRNAs containing short 5' leaders and 3' trailers; however, leader and trailer lengths do not significantly alter the observed rate constants of PRORP2 in single-turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates., (Published by Elsevier Ltd.)

Previous molecular dynamics (MD) simulations of ion-lipid interactions have focused on pure phospholipid bilayers. Many functional microdomains in membranes have a complex composition of cholesterol and phospholipids. Here, we reveal the distinctiveness of the interactions and the effects of the ions on a cholesterol-rich bilayer by performing MD simulations of a cholesterol-rich bilayer with a Na(+)/K(+) mixture or a Na(+)/K(+)/Ca(2+)/Mg(2+) mixture. The simulations reveal that Ca(2+) maintains its dominant role in the interaction with the cholesterol-rich bilayer, but the binding affinity of Mg(2+) to the cholesterol-rich bilayer is even weaker than the affinities of Na(+) and K(+), whereas its interaction with pure phospholipid bilayers is strong and is only slightly weaker than that of Ca(2+). Additionally, it was found that the presence of additional divalent cations induces the headgroups of phospholipids to be more perpendicular to the membrane surface, reducing the lateral movement of lipids and slightly altering the ordering and packing of the cholesterol-rich bilayer, different from divalent cations, which strongly influence that ordering and packing of pure phospholipid bilayers. Therefore, this study indicates that cholesterol in the membrane could affect the interactions between membrane and cations. The findings could be helpful in understanding the biological processes relevant to regulation of cations in cholesterol-rich regions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations of Gln41 and Lys75 with nonpolar residues in the N-terminal domain of calmodulin (N-Cam) revealed the importance of solvation energetics in conformational change of Ca(2+) sensor EF-hand domains. While in general these domains have polar residues at these corresponding positions yet the extent of their conformational response to Ca(2+) binding and their Ca(2+) binding affinity can be different from N-Cam. Consequently, here we address the charge state of the polar residues at these positions. The results show that the charge state of these polar residues can affect substantially the conformational change and the Ca(2+) binding affinity of our N-Cam variants. Since all the variants kept their conformational activity in the presence of Ca(2+) suggests that the differences observed among them mainly originate from the difference in their molecular dynamics. Hence we propose that the molecular dynamics of Ca(2+) sensor EF-hand domains is a key factor in the multifunctional aspect of EF-hand proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and proteins. One of the main partner-regulator of VDAC is the 18 kDa translocator protein (TSPO), whose role in the regulation of membrane permeability is not completely understood. We show that TSPO ligands, 1 μM PPIX and PK11195 at concentrations of 50 μM, accelerate opening of permeability transition pores (mPTP) in Ca(2+)-overloaded rat brain mitochondria (RBM). By contrast, PK11195 at 100 nM and anti-TSPO antibodies suppressed pore opening. Participation of VDAC in these processes was demonstrated by blocking VDAC with G3139, an 18-mer phosphorothioate oligonucleotides, which sensitized mitochondria to Ca(2+)-induced mPTP opening. Despite the inhibitory effect of 100 nM PK11195 and anti-TSPO antibodies alone, their combination with G3139 considerably stimulated the mPTP opening. Thus, 100 nM PK11195 and anti-TSPO antibody can modify permeability of the VDAC channel and mPTP. When VDAC channels are closed and TSPO is blocked, permeability of the VDAC for calcium seems to be the highest, which leads to accelerated pore opening., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Guanine-rich sequences can, under appropriate conditions, adopt a distinctive, four-stranded, helical fold known as a G-quadruplex. Interest in quadruplex folds has grown in recent years as evidence of their biological relevance has accumulated from both sequence analysis and function-specific assays. The folds are unusually stable and their formation appears to require close management to maintain cell health; regulatory failure correlates with genomic instability and a number of cancer phenotypes. Biologically relevant quadruplex folds are anticipated to form transiently in mRNA and in single-stranded, unwound DNA. To elucidate factors, including bound solvent, that contribute to the stability of RNA quadruplexes, we examine, by X-ray crystallography and small-angle X-ray scattering, the structure of a previously reported tetramolecular quadruplex, UGGGGU stabilized by Sr(2+) ions. Crystal forms of the octameric assembly formed by this sequence exhibit unusually strong diffraction and anomalous signal enabling the construction of reliable models to a resolution of 0.88Å. The solvent structure confirms hydration patterns reported for other nucleic acid helical conformations and provides support for the greater stability of RNA quadruplexes relative to DNA. Novel features detected in the octameric RNA assembly include a new crystal form, evidence of multiple conformations and structural variations in the 3' U tetrad, including one that leads to the formation of a hydrated internal cavity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X(2+))-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X(2+) binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca(2+) and Mg(2+) binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca(2+) sites resulted in greater compaction than for Mg(2+) alone where this effect was additive or greater when both ions were present at physiological levels. Less X(2+)-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X(2+) sites of the Gla domain and higher avidity X(2+) sites of the epidermal growth factor 1 (EGF1)-like domain., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To identify the fuctions of the nine putative O-methyltransferase genes in Streptomyces virginiae IBL14, the evolutionary and functional relationship of these genes in its 8.0 Mb linear chromosome was set up via sequence comparison with those of other Streptomyces species. Further, the functions and catalytic mechanism of the three genes sviOMT02, sviOMT03 and sviOMT06 from this strain were studied through experimental and computational approaches. As a result, the nine putative O-methyltransferases belong to methyltransf_2 superfamily, amdomet-MTases superfamily, and leucine carboxyl methyltransferase superfamily, and are phylogenetically close to those of Streptomyces sp. C. The products of genes sviOMT03 and sviOMT06 could catalyze O-methylation of caffeic acid to form ferulic acid. Computational analysis indicated that the O-methylation mechanism of SviOMT03 and SviOMT06 proceeds from a direct transfer of the SAM-methyl group to caffeic acid with inversion of symmetry aided by a divalent metal ion in a SN2-like mechanism. Particularly, the conservative polar amino acid residues in SviOMT03 and SviOMT06, including Lys143 that reacts with caffeic acid, Ser74, Asp140 and Tyr149 that react with S-adenosyl methionine, and His142 (SviOMT03) or His171 (SviOMT06) that transfers the 3-hydroxyl proton of substrate caffeic acid, probably be essential in their O-methylation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

High pressure thermal (HPT) processing can be used to improve traditional preservation methods and increase food safety and durability, whereas quality related characteristics can be largely maintained. Clostridium (C.) botulinum type E is a non-proteolytic, psychrotrophic, toxin-producing spore former, commonly associated with aquatic environments in temperate regions of the northern hemisphere. Sporulation in nature is likely to occur under varying conditions including temperature and nutrient availability, which might affect resistance properties of resulting spores. In our study, we determined the effect of sporulation temperature (13-38 °C) on the resistance of three Clostridium botulinum type E strains to differently intense HPT treatments (200 MPa at 40 and 80 °C, and 800 MPa at 40 and 80 °C). Furthermore, the effect of cations on sporulation temperature-mediated alterations in HHP resistance was investigated. Results indicate that low and high sporulation temperatures can increase and decrease sporal HPT resistance, respectively, in a treatment-dependent (pressure level, treatment temperature) manner, whereas the trends observed are largely unaffected by pressure dwells (1 s-10 min). Furthermore, results show that the cation content of the sporulation medium (Ca(2+), Mg(2+), Mn(2+)) marginally influences and partially counteracts effects on the HPT resistance of spores grown at low and elevated temperatures, respectively. This suggests that sporulation temperature and medium cations provoke changes in some common spore resistance structures. Sporulation conditions can markedly affect spore resistance properties and, thus, should be considered for the experimental setup of worst case studies aiming to evaluate the effectiveness of food processes in terms of the inactivation of C. botulinum type E spores., (Copyright © 2014 Elsevier Ltd. All rights reserved.)