Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.     

Purification and Characterization of a Human Brain Galectin-1 Ligand

Abstract: Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N -linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.     

Genetic Analysis of the Biosynthesis of 2-Methoxy-3-Isobutylpyrazine,a Major Grape-Derived Aroma Compound Impacting Wine Quality

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.The pleasure experienced while enjoying a glass of wine is the result of sophisticated sensory, neurophysiological, and psychological processes triggered by wine aroma. Wine flavor is the result of a complex mixture of volatile compounds in the headspace of the glass that induces feelings of pleasure at the brain level (Shepherd, 2006). During the last 40 years, over 800 volatile molecules have been formally identified in wines, in concentrations ranging from hundreds of milligrams per liter down to a few picograms per liter (Ebeler and Thorngate, 2009; Styger et al., 2011). Among all of them, a relatively limited number of compounds, called varietal (or primary) aromas, play a crucial role in wine flavor and typicality. These aromas, which are related to the grape variety, belong to a limited number of chemical families, including monoterpenes, C13 norisoprenoids, volatile sulfur compounds, and methoxypyrazines (MPs; Ebeler and Thorngate, 2009). Quite frequently, they exist mostly in the grape (Vitis vinifera) berry as nonvolatile, odorless, “bound” forms that can be released by chemical and enzymatic reactions occurring during the winemaking and wine aging processes, thus enhancing wine’s varietal expression (Styger et al., 2011). Two classical examples are the glycoside precursors of the monoterpenols (Strauss et al., 1986) and the cysteinylated or glutathionylated precursors of the volatile thiols (Tominaga et al., 1998; Peña-Gallego et al., 2012). Noticeable exceptions are the MPs, which are found in grape berries exclusively as free, volatile molecules.MPs are strongly odorant volatile heterocycles, with vegetable-like fragrances, that are widely occurring in the plant kingdom (Maga, 1982). In grape, they can be detected in fruits, leaves, shoots, and roots (Dunlevy et al., 2010). They are found in different grape varieties and are particularly abundant in the so-called Bordeaux cultivars (i.e. cv Cabernet Franc, Cabernet Sauvignon [CS], Sauvignon Blanc, Merlot, and Carménère [Car]; Bayonove et al., 1975; Lacey et al., 1991; Roujou de Boubée et al., 2002; Belancic and Agosin, 2007), whereas they are rarely detected in other cultivars, such as cv Pinot Noir (PN), Chardonnay, or Petit Verdot (PV). This finding indicates a strong genotype dependency of MP biosynthesis (Koch et al., 2010). MPs are accumulated in berries until bunch closure or véraison, and then their level declines after véraison (Hashizume and Samuta, 1999; Ryona et al., 2008). MP concentration in wine is highly correlated with the grape berry content at harvest (Roujou de Boubée et al., 2002). Three MPs are found in grape berries: 2-methoxy-3-isobutylpyrazine (IBMP), which is the most abundant, and two others, 2-methoxy-3-isopropylpyrazine (IPMP) and 2-methoxy-3-sec-butylpyrazine (SBMP; Ebeler and Thorngate, 2009). Both IBMP and IPMP display very low sensory detection thresholds in the wine matrix, ranging from 1 to 16 ng L^–1.MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols (Dubourdieu et al., 2006; Lund et al., 2009). Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these extremely powerful odorants will reduce consumer acceptance (Parr et al., 2007). In red wine, MPs are considered as off-flavor, and red wines can be depreciated by concentrations above 10 ng L^–1 (Allen et al., 1991; Roujou de Boubée et al., 2000; Belancic and Agosin, 2007). Given the importance of MPs, either as typical varietal aromas or as detrimental off-flavors, deciphering the genetic and molecular determinism of their accumulation is of high interest for viticulture.In spite of this, until recently little was known about the MP biosynthesis pathway or the MP biosynthetic genes, either in grapevine or other plant species. Theoretical biosynthesis pathways have been proposed since the mid-1970s. They all start by the addition of an α-dicarbonyl on a branched amino acid (Leu for IBMP, Val for IPMP) to form a 2-hydroxy-3-alkylpyrazine, which is subsequently transformed into the corresponding MP, by a methoxylation reaction (Murray and Whitfield 1975; Gallois et al., 1988). While the initial addition step remains to be demonstrated in plants, an S-adenosyl-l-Met (SAM)-dependent O-methyltransferase (OMT), capable of converting 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP, has been detected in CS shoots, partially purified and sequenced (Hashizume et al., 2001a, 2001b; Fig. 1). Recently, Dunlevy et al. (2010) characterized two OMTs, VvOMT1 and VvOMT2, capable of methylating IBHP in vitro, albeit with high apparent K_m values. To investigate the genetic bases of MP biosynthesis in grape berries, we performed a quantitative trait loci (QTL) analysis, which has led to the identification of two previously uncharacterized OMTs termed VvOMT3 and VvOMT4. Functional characterization of these two OMTs showed that VvOMT3 was highly specific and efficient for IBHP methylation. Based on its differential expression in high-MP and low-MP grapevine varieties, we propose that VvOMT3 and, to a lesser extent, VvOMT4 are key genes for MP biosynthesis in grapevine berries.Open in a separate windowFigure 1.Putative biosynthesis pathway for IBMP adapted from Hashizume et al. (2001a). SAHcy, S-Adenosyl-l-homo-Cys.     

Phosphorylation of Parkin by the cyclin-dependent kinase 5 at the linker region modulates its ubiquitin-ligase activity and aggregation

Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/alpha-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease.     

Three novel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 isoforms. Expression, regulation, and function of the membranes of the SERCA3 family

Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a-c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a-e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.     

Proteomic analysis of a lymphoma-derived cell line (DG75) following treatment with a demethylating drug: modification of membrane-associated proteins

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.     

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.     

Spatial pattern of central African rainforests can be predicted from average tree size

When considering all trees irrespective of their species, natural tropical rain forests typically exhibit spatial patterns that range from random to regular. The regularity is often interpreted as a footprint of tree competition. Using 23 permanent sample plots totalling 61 ha in the rain forests of central Africa, we characterized their spatial patterns and modelled those that exhibited regularity by a Strauss point process. This Strauss process is obtained as a Markov point process whose interaction function is an exponential function of a competition index commonly used in forestry. The parameter of this Strauss process characterizes the strength of competition. The 23 plots in central Africa differed in tree density and basal area, and could be discriminated depending on the type of spatial patterns: plots having a large basal area with respect to their density had a non regular pattern, whereas those having a small basal area with respect to their density had a regular pattern. For those plots that exhibited regularity, average tree size could be used to predict the strength of competition. The parameter of the Strauss process was significantly related to the average size by a linear relationship, such that competition decreases as average tree size increases. This relationship extrapolated to a null value of the Strauss parameter when average tree size reaches 32 cm in diameter. This relationship between average tree size and spatial pattern is a testable feature for future studies on the relationship between competition and spatial pattern in natural forests.     

Application of metal-chelate affinity chromatography to the study of the phosphoproteome

Summary. With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe³⁺ was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed.     

Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread.