Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

Phospholipase Cdelta1 associates with importin beta1 and translocates into the nucleus in a Ca2+-dependent manner

Phospholipase C (PLC)delta1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCdelta1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCdelta1 binds with importins. PLCdelta1 interacted with importin beta1 in a Ca2+-dependent manner in vitro even in the absence of importin alpha. A PLCdelta1 mutant E341A, which lacks Ca2+-binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCdelta1 is mediated by its Ca2+-dependent interaction with importin beta1.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Effects of Al3+ on Ca2+-ATPase Activity on Chloroplasts of Rice and Relation to Calmodulin

The relationship between aluminium phytotoxicity and calmodulin has been studied with. calcium-dependent ATPase in chloroplasts of rice. This enzyme could be activated by extrinsic calmodulin. It showed that the activity of Ca2+-ATPase in chloroplasts was regulated by calmodulin. The activation of calmodulin to the enzyme might be inhibited by calmodulin antagonists, TFP and CPZ. The effects of A13+ on the activation of calmodulin was similar to that of the calmodulin antagonists. Calcium could reduce the inhibition of aluminiutn. It seems that there is a model of toxic responses in plants to aluminium: Al3+→calmodulin→target enzy mes→metabolism.     

Acute effect of beta amyloid on synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks

The effects of beta amyloid (Abeta) on cytoplasmic Ca(2+) ([Ca(2+)](c)) have been studied extensively, but the current literature on this aspect is confusing. We reported that 20 microM Abeta(25-35) significantly inhibited the synchronized spontaneous cytoplasmic Ca(2+) transients immediately after application, whereas it had little effect on the baseline [Ca(2+)](c) concentration in neurons. Abeta(1-42) had a similar effect on the Ca(2+) transients as Abeta(25-35), while it increased baseline [Ca(2+)](c) concentration gradually. However, Abeta(1-40) had little effect on either Ca(2+) transients or baseline [Ca(2+)](c). Such differential effects of Abeta on Ca(2+) signals might explain, at least partially, the confusing observations from the previous studies and provide important therapeutic implications for preventing or reversing early neuron damage in Alzheimer's disease.     

Distinctive Conformation of Minor Site‐Specific Nuclear Localization Signals Bound to Importin‐α

Nuclear localization signals (NLSs) contain one or two clusters of basic residues and are recognized by the import receptor importin‐α. There are two NLS‐binding sites (major and minor) on importin‐α and the major NLS‐binding site is considered to be the primary binding site. Here, we used crystallographic and biochemical methods to investigate the binding between importin‐α and predicted ‘minor site‐specific’ NLSs: four peptide library‐derived peptides, and the NLS from mouse RNA helicase II/Guα. The crystal structures reveal that these atypical NLSs indeed preferentially bind to the minor NLS‐binding site. Unlike previously characterized NLSs, the C‐terminal residues of these NLSs form an α‐helical turn, stabilized by internal H‐bond and cation‐π interactions between the aromatic residues from the NLSs and the positively charged residues from importin‐α. This helical turn sterically hinders binding at the major NLS‐binding site, explaining the minor‐site preference. Our data suggest the sequence RXXKR[K/X][F/Y/W]XXAF as the optimal minor NLS‐binding site‐specific motif, which may help identify novel proteins with atypical NLSs .     

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina

Neurotransmitters affect neuronal development by regulating intracellular Ca²⁺ concentrations. We studied spatiotemporal pattern of the development of glutamate-induced intracellular Ca²⁺ rise in the embryonic chick retina, where developmental changes in mitotic activity, cell death, and synapse formation have been well established. Glutamate was bath-applied to the central part of the retina dissected at embryonic day 3 (E3) to E13, and changes in intracellular Ca²⁺ concentration were measured with Fura-2 fluorescence. The Ca²⁺ rise to glutamate first appeared at E6, reached a maximum at E9–10, and then declined before the appearance of synaptic structures (E12). Ca²⁺ rises to kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) appeared earlier and were larger in amplitude than those to N-methyl-D -aspartic acid. The KA/AMPA receptor of the E9 chick retina was permeable for Ca²⁺, suggesting the functional expression of Ca²⁺-permeable KA/AMPA receptors at the stage of retinal cell death. The Ca²⁺rise to glutamate and KA occurred intensely at the inner plexiform layer, the inner part of inner nuclear layer, and the ganglion cell layer, where the cell death occurs. The Ca²⁺ rise to high K²⁺, in contrast, occurred intensely at the nerve fiber layer and the ganglion cell layer, developing continuously from E3 until E11. Our study shows that the Ca²⁺ rise to glutamate develops with the decline of the mitotic activity of the retinal cells and is transiently enhanced during the period of cell death in the embryonic chick retina. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 113–125, 1998     

PGF2alpha increases FGF-2 and FGFR2 trafficking in Py1a rat osteoblasts via clathrin independent and importin beta dependent pathway

Previous studies showed that prostaglandin F2alpha (PGF2alpha) stimulated fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2alpha increased the formation of clathrin-coated structures in Py1a rat osteoblasts, they were not involved in FGF-2 and FGFR2 trafficking. PGF2alpha increased binding of FGF-2 and FGFR2 and co-localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF-2 and FGFR2 were in close spatial correlation with importin beta, further supporting nuclear import of the FGF-2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin beta protein labeling in response to PGF2alpha. Similar to PGF2alpha, phorbol 12-myristate 13-acetate (PMA) also increased importin beta protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF-2/FGFR2/importin beta nuclear trafficking.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt,Cynops pyrrhogaster

Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca²⁺ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca²⁺ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca²⁺ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca²⁺ channels mediate the intracellular Ca²⁺ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.     

重组PICK1蛋白的多聚形式及其与Ca2+和Mg2+的结合

蛋白激酶Cα相互作用蛋白1(PICK1) 是从线虫到人的所有生物中非常保守的一类存在于细胞质中的膜结合蛋白,在蛋白质转运,以及细胞内信号转导过程中发挥重要作用.通过基因重组技术获得PICK1及其截短的 N-PDZ(1～110 残基)和 BAR-C(128～416残基)重组蛋白,结合变性与非变性聚丙烯酰胺凝胶电泳,以及分子排阻层析,表明溶液中的PICK1主要以二聚体形式存在.利用荧光光谱分析PICK1与金属离子Ca²⁺和Mg²⁺的结合情况.结果表明,在0.02 mol/L Hepes, pH 7.2,随着2种金属离子的不断滴加,PICK1在338 nm 处的最大荧光强度逐渐降低,PICK1与Ca²⁺结合常数为Ka1=（2.34±0.20）×10.6 L/mol^-1,Ka2=(7.75±0.62)×10.5 L/mol^-1,而Mg²⁺结合常数为Ka=（5.00±0.40）×10.6 L/mol^-1.另外,对PICK1的N端区域N-PDZ和C端区域BAR-C的重组片段与金属离子Ca²⁺和Mg²⁺结合情况进一步分析表明,Ca²⁺既能与PICK1的N 端N-PDZ结合,又可与C端BARC结合,而Mg²⁺只结合在PICK1的N-PDZ区域.比较Ca²⁺或Mg²⁺对PICK1结合脂质的影响,显示Ca²⁺能明显增强蛋白和脂质的结合.     

Structures of EF-hand Ca 2+-binding proteins: Diversity in the organization, packing and response to Ca 2+ Binding

The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the coformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.© Kluwer Academic Publishers     

Structural characterization and optical properties of Ca2MgSi2O7:Eu2+,Dy3+ phosphor by solid‐state reaction method

Ca₂MgSi₂O₇:Eu²⁺,Dy³⁺ phosphor was prepared by the solid‐state reaction method under a weak reducing atmosphere. The obtained phosphor was characterized using X‐ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), energy dispersive X‐ray spectroscopy (EDX) and Fourier transform infrared (FT‐IR) techniques. The phase structure of the Ca₂MgSi₂O₇:Eu²⁺,Dy³⁺ phosphor was akermanite type, which is a member of the melilite group. The surface morphology of the sintered phosphor was not uniform and phosphors aggregated tightly. EDX and FT‐IR spectra confirm the elements present in the Ca₂MgSi₂O₇:Eu²⁺,Dy³⁺ phosphor. Under UV excitation, a broadband emission spectrum was found. The emission spectra observed in the green region centered at 535 nm, which is due to the 4f–5d transition. The mechanoluminescence (ML) intensity of the prepared phosphor increased linearly with increases in the mechanical load. The ML spectra were similar to the photoluminescence (PL), which indicates that ML is emitted from the same emitting center of Eu²⁺ ions as PL. Copyright © 2014 John Wiley & Sons, Ltd.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

A novel red phosphor Ca2YNbO6:Eu3+ for WLEDs

Due to the advantages of good physicochemical properties, thermal stability, and optical properties, double perovskite compounds have received extensive attention. On this basis, a new type of red phosphor, Ca₂YNbO₆:xEu³⁺, was synthesized using a high-temperature solid-phase method. Its phase purity, morphology, elemental composition, absorption spectrum, photoluminescence, thermal stability, and Commission Internationale de l'éclairage (CIE) chromaticity coordinates were thoroughly investigated. The results display that there is no impurity phase in the samples and the convergence factor R_wp = 14.2%; the microscopic particles are uniform and full, and the distribution of each element is uniform. The energy band gap ΔE is between 3.71 eV and 3.65 eV. The luminescence intensity is the best when the doped Eu³⁺ concentration x reaches 0.4, and emits 612 nm red light (⁵D₀→⁷F₂) under 465 nm excitation, and the concentration quenching is attributed to a d–d interaction. The luminescence intensity at 425 K was still 75% of the room temperature luminescence intensity, which indicates that the thermal stability is extremely superior. The CIE chromaticity coordinates (0.6534, 0.3455) of the Ca2YNbO6:0.4Eu³⁺ phosphors are very close to National Television Standards Committee (0.670, 0.330), and the samples have low correlated colour temperature (2656 K) and high colour purity (99.90%). All findings suggest that Ca₂YNbO₆:Eu³⁺ can serve as a substitute for red phosphor in WLEDs.     

Choline Is an Intracellular Messenger Linking Extracellular Stimuli to IP3-Evoked Ca2+ Signals through Sigma-1 Receptors

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