A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.     

Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells

Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase-anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.     

Photoperiodic flowering occurs under internal and external coincidence

Determining the proper time to flower is important to ensure the reproductive success of plants. The model plant Arabidopsis is able to measure day-length and promotes flowering in long day (LD) conditions. One of the most prominent mechanisms in photoperiodic flowering is the clock-regulated gene expression of CONSTANS (CO) and the stabilization and activation of CO protein by light (regarded as external coincidence). We recently demonstrated that timing of the blue-light dependent formation of FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and GIGANTEA (GI) protein complex is crucial for regulating the timing of CO gene expression. The expression of FKF1 and GI is clock regulated, and their expression patterns have the same phase in LD (regarded as internal coincidence) but not in short day (SD) conditions, where floral induction is greatly delayed. Hence, timing of the FKF1-GI complex formation is regulated by the coincidence of both external and internal cues. Here, we propose a molecular mechanism for CO regulation by FKF1-GI complex formation.Key words: Arabidopsis, circadian clock, photoperiodic flowering, CONSTANS, GIGANTEA, FKF1, CDF1     

Essential role of EP3 subtype in prostaglandin E2-induced adhesion of mouse cultured and peritoneal mast cells to the Arg-Gly-Asp-enriched matrix

Accumulating evidence has indicated that mast cells can modulate a wide variety of immune responses. Migration and adhesion play a critical role in regulation of tissue mast cell function, in particular, under inflammatory conditions. We previously demonstrated that prostaglandin (PG) E(2) stimulates adhesion of a mouse mastocytoma cell line, P-815, to the Arg-Gly-Asp (RGD)-enriched matrix through cooperation between two PGE(2) receptor subtypes: EP3 and EP4 (Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. J Biol Chem 278: 17977-17981, 2003). We here investigated PGE(2)-induced adhesion of IL-3-dependent bone marrow-derived cultured mast cells (BMMCs). In contrast to the elevated cAMP-dependent adhesion of P-815 cells, EP3-mediated Ca(2+) mobilization plays a pivotal role in PGE(2)-induced adhesion of BMMCs. Adhesion and Ca(2+) mobilization induced by PGE(2) were abolished in the Ptger3(-/-) BMMCs and were significantly suppressed by treatment with pertussis toxin, a phospholipase C inhibitor, U-73122, and a store-operated Ca(2+) channel inhibitor, SKF 36965, indicating the involvement of G(i)-mediated Ca(2+) influx. We then investigated PGE(2)-induced adhesion of peritoneal mast cells to the RGD-enriched matrix. EP3 subtype was found to be the dominant PGE receptor that expresses in mouse peritoneal mast cells. PGE(2) induced adhesion of the peritoneal mast cells of the Ptger3(+/+) mice, but not that of the Ptger3(-/-) mice. In rat peritoneal mast cells, PGE(2) or an EP3 agonist stimulated both Ca(2+) mobilization and adhesion to the RGD-enriched matrix. These results suggested that the EP3 subtype plays a pivotal role in PGE(2)-induced adhesion of murine mast cells to the RGD-enriched matrix through Ca(2+) mobilization.     

A model for synaptic development regulated by NMDA receptor subunit expression

Activation of NMDA receptors (NMDARs) is highly involved in the potentiation and depression of synaptic transmission. NMDARs comprise NR1 and NR2B subunits in the neonatal forebrain, while the expression of NR2A subunit is increased over time, leading to shortening of NMDAR-mediated synaptic currents. It has been suggested that the developmental switch in the NMDAR subunit composition regulates synaptic plasticity, but its physiological role remains unclear. In this study, we examine the effects of the NMDAR subunit switch on the spike-timing-dependent plasticity and the synaptic weight dynamics and demonstrate that the subunit switch contributes to inducing two consecutive processes—the potentiation of weak synapses and the induction of the competition between them—at an adequately rapid rate. Regulation of NMDAR subunit expression can be considered as a mechanism that promotes rapid and stable growth of immature synapses. Action Editor: Upinder Bhalla     

Molecular chaperone activity of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) endoplasmic reticulum-located small heat shock protein

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His₆-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.