Protein Tyrosine Phosphatase 4A3 (PTP4A3) Is Required for Xenopus laevis Cranial Neural Crest Migration In Vivo

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.     

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

Identification of Mitochondrial Coenzyme A Transporters from Maize and Arabidopsis

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.CoA acts as an acyl carrier in many reactions of primary and secondary metabolism, and some 8% of the nearly 4,900 enzymes described in the Enzyme Commission database are CoA dependent (Bairoch, 2000). CoA occupies a central position in lipid metabolism, respiration, gluconeogenesis, and other pathways (Leonardi et al., 2005). It is present in all forms of life, but while all organisms can synthesize it from pantothenate (vitamin B₅), only prokaryotes, plants, and fungi are able to synthesize pantothenate; animals obtain pantothenate from the diet (Daugherty et al., 2002; Leonardi et al., 2005; Webb and Smith, 2011).In plants, the steps that convert pantothenate to CoA are almost certainly cytosolic (Webb and Smith, 2011; Gerdes et al., 2012). CoA, however, is required in mitochondria for the citric acid cycle, in chloroplasts for fatty acid synthesis, and in peroxisomes for β-oxidation. CoA, therefore, must be imported into these organelles from the cytosol, and indeed, early work demonstrated a CoA transport system in potato (Solanum tuberosum) mitochondria (Neuburger et al., 1984). Yeast (Saccharomyces cerevisiae) and mammalian mitochondria and peroxisomes likewise import CoA because they cannot make it (Fiermonte et al., 2009; Agrimi et al., 2012b). The compartmentation of CoA in all eukaryotes appears to be closely regulated, with cytosol and organelles maintaining separate CoA pools whose levels can modulate fluxes through CoA-dependent reactions (Hunt and Alexson, 2002; Leonardi et al., 2005; De Marcos Lousa et al., 2013).Mitochondrial CoA transporters belonging to the mitochondrial carrier family (MCF) have been identified in yeast (Leu-5p; Prohl et al., 2001) and human (SLC25A42; Fiermonte et al., 2009). Furthermore, peroxisomal CoA carriers from human (SLC25A17; Agrimi et al., 2012b) and Arabidopsis (Arabidopsis thaliana; peroxisomal CoA and NAD carrier [PXN]; Agrimi et al., 2012a) have also been identified. However, no transporters for CoA are known for plant mitochondria or chloroplasts (Palmieri et al., 2011; Gerdes et al., 2012).In this study, a comparative genomic analysis first identified close Arabidopsis and maize (Zea mays) homologs of the yeast and mammalian mitochondrial CoA carriers as candidates for the missing plant mitochondrial or chloroplast transporters. Experimental evidence then demonstrated that the candidate proteins transport CoA when expressed in yeast, that they are targeted to mitochondria in vitro and in planta, and that they are expressed throughout the plant.     

Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus Magnaporthe oryzae

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.     

Identification of calcitonin expression in the chicken ovary: influence of follicular maturation and ovarian steroids

Calcitonin (CALCA), a hormone primarily known for its role in calcium homeostasis, has recently been linked to reproduction, specifically as a marker for embryo implantation in the uterus. Although CALCA expression has been documented in several tissues, there has been no report of production of CALCA in the ovary of any vertebrate species. We hypothesized that the Calca gene is expressed in the chicken ovary, and its expression will be altered by follicular maturation or gonadal steroid administration. Using RT-PCR, we detected Calca mRNA and the calcitonin receptor (Calcr) mRNA in the granulosa and theca layers of preovulatory and prehierarchial follicles. Both CALCA and Calca mRNA were localized in granulosa and thecal cells by confocal microscopy. Using quantitative PCR analysis, F1 follicle granulosa layer was found to contain significantly greater Calca mRNA and Calcr mRNA levels compared with those of any other preovulatory or prehierarchial follicle. The granulosa layer contained relatively greater Calca and Calcr mRNA levels compared with the thecal layer in both prehierarchial and preovulatory follicles. Progesterone (P(4)) treatment of sexually immature chickens resulted in a significantly greater abundance of ovarian Calca mRNA, whereas estradiol (E(2)) or P(4) + E(2) treatment significantly reduced ovarian Calca mRNA quantity. Treatment of prehierarchial follicular granulosa cells in vitro with CALCA significantly decreased FSH-stimulated cellular viability. Collectively, our results indicate that follicular maturation and gonadal steroids influence Calca and Calcr gene expression in the chicken ovary. We conclude that ovarian CALCA is possibly involved in regulating follicular maturation in the chicken ovary.     

Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the Mediterranean Sea in relation to environmental variables

Aerobic anoxygenic phototrophic bacteria (AAP) represent an important fraction of bacterioplankton assemblages in various oceanic regimes. Although their abundance and distribution have been explored recently in diverse oceanic regions, the environmental factors controlling the population structure and diversity of these photoheterotrophic bacteria remain poorly understood. Here, we investigate the horizontal and vertical distributions and the genetic diversity of AAP populations collected in late summer throughout the Mediterranean Sea using pufM-temporal temperature gel gradient electrophoresis (TTGE) and clone library analyses. The TTGE profiles and clone libraries analyzed using multivariate statistical methods demonstrated a horizontal and vertical zonation of AAP assemblages. Physicochemical parameters such as pH, inorganic nitrogen compounds, photosynthetically active radiation, total organic carbon and to a lesser extent particulate organic nitrogen and phosphorus, and biogenic activities (e.g. bacterial production, cell densities), acted in synergy to explain the population changes with depth. About half of the pufM sequences were <94% identical to known sequences. The AAP populations were predominantly (~80%) composed of Gammaproteobacteria, unlike most previously explored marine systems. Our results suggest that genetically distinct ecotypes inhabiting different niches may exist in natural AAP populations of the Mediterranean Sea whose genetic diversity is typical of oligotrophic environments.