Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.     

(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1

The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C(4) and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy.     

Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.     

Docosahexaenoic acid down-regulates endothelial Nox 4 through a sPLA2 signalling pathway

We investigated the anti-inflammatory and antioxidant activities of docosahexaenoic acid (DHA) by evaluating its modulation of the two enzymes most involved in vascular inflammation, i.e. endothelial secreted phospholipase A₂ (sPLA₂) and NADPH oxidase 4 (Nox) 4. Exposure of human aortic endothelial cells (HAECs) to DHA led to its preferential incorporation into outer leaflet phospholipids. Pre-treatment with DHA abolished HAECs stimulation induced by A23187 and Ang II, whereas the effects on IL-1β treatment were less pronounced. Group V sPLA₂ RNA was similarly modulated by DHA supplementation. In addition, DHA decreased Nox 4 expression and activity; this effect was associated with reduced production of reactive oxygen species. Further, the use of specific inhibitors allowed demonstrating that group V sPLA₂ is involved in the down-regulation of Nox 4 expression and activity by DHA. This interplay is mediated by ERK and PKC.     

Transition from dimers to higher oligomeric forms occurs during the ATPase cycle of the ABCA1 transporter

Fluorescence resonance energy transfer and native PAGE analytical techniques were employed to assess the quaternary structure of ABCA1, an ATP binding cassette transporter playing a crucial role in cellular lipid handling. These experimental approaches support the conclusion that ABCA1 is associated in dimeric structures that undergo transition into higher order structures, i.e. tetramers, during the ATP catalytic cycle. Our data hence underline molecular assembly as a crucial parameter in ABCA1 function and the advantage of native PAGE as analytical tool for intractable membrane proteins.     

Investigation of noncalcium interactions of fura-2 by classical and synchronous fluorescence spectroscopy.

Several authors have reported unexpected intracellular spectra of both indo-1 and fura-2. One of the major methodological problems in the evaluation of calcium concentration using fluorescent probes is that it is assumed that only two forms of the dyes are detectable within the cells. We show in this study of fura-2 properties that this calcium probe is pH-sensitive and able to bind to cellular proteins. The excitation spectra of protonated and protein-bound forms of fura-2 exhibit a maximum in the same region as that associated with the calcium-free form (i.e., near 365 nm). The very small shift in the excitation spectra upon proton or protein binding precludes the use of classical methods to determine the spectral composition of mixtures of several forms of fura-2. We therefore used the synchronous fluorescence technique to detect the protein-bound form of fura-2 selectively, in order to assess the pH dependence of the fura-2/protein interaction. The nonspecific binding of fura-2 to proteins is reinforced at acidic pH and inhibited by calcium. The fact that the same type of interaction was found between fura-2 and poly-L-lysine suggests that it could be mediated by basic amino acids. Because of the strong overlap of the excitation spectrum of the unprotonated free fura-2 with those associated with the protonated and protein-bound forms, a cytoplasmic acidification may lead to an artifactual measurement of low calcium levels.     

KIT Mutation and Loss of 14q May Be Sufficient for the Development of Clinically Symptomatic Very Low-Risk GIST

The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.     

Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.Plants have evolved a complex array of defenses when attacked by microbial pathogens. The success of plant resistance first relies on the capacity of the plant to recognize its invader. Among early events, a transient production of reactive oxygen species (ROS), known as the oxidative burst, is characteristic of successful pathogen recognition (Torres, 2010). Perception of pathogen attack then initiates a large array of immune responses, including modification of cell walls, as well as the production of antimicrobial proteins and metabolites like pathogenesis-related (PR) proteins and phytoalexins, respectively (Schwessinger and Ronald, 2012). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are key players in the signaling networks involved in plant resistance (Bari and Jones, 2009; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate array of defense responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). Whereas SA is considered essential for resistance to (hemi)biotrophic pathogens, it is assumed that JA and ET signaling pathways are important for resistance to necrotrophic pathogens in Arabidopsis (Arabidopsis thaliana; Thomma et al., 2001; Glazebrook, 2005). A successful innate immune response often includes the so-called hypersensitive response (HR), a form of rapid programmed cell death (PCD) occurring in a limited area at the site of infection. This suicide of infected cells is thought to limit the spread of biotrophic pathogens, including viruses, bacteria, fungi, and oomycetes (Mur et al., 2008).During the past decade, significant progress has been made in our understanding of the cellular function of plant sphingolipids. Besides being structural components of cell membranes, sphingolipids are bioactive metabolites that regulate important cellular processes such as cell survival and PCD, occurring during either plant development or plant defense (Dunn et al., 2004; Berkey et al., 2012; Markham et al., 2013). The first evidence of the role of sphingolipids in these processes came from the use of the fungal toxins fumonisin B1 (FB1) and AAL, produced by the necrotrophic agent Alternaria alternata f. sp. lycopersici. These toxins are structural sphingosine (d18:1) analogs and function as ceramide synthase inhibitors. They triggered PCD when exogenously applied to plants. Mutant strains in which the production of such toxins is abrogated failed to infect the host plant, implying that toxin accumulation is required for pathogenicity and that the induction of plant PCD could be considered a virulence tool used by necrotrophic pathogens (Berkey et al., 2012). Moreover, several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD in plants. For example, exogenously applied Cers and LCBs (d18:0, d18:1, or t18:0) induced PCD either in cell suspension cultures (Liang et al., 2003; Lachaud et al., 2010, 2011; Alden et al., 2011) or in whole seedlings (Shi et al., 2007; Takahashi et al., 2009; Saucedo-García et al., 2011). AAL- and FB1-induced PCD seemed to be due to the accumulation of free sphingoid bases (dihydrosphingosine [d18:0] and phytosphingosine [t18:0]; Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007). Spontaneous cell death in lag one homolog1 or l-myoinositol1-phosphate synthase mutant could be due to trihydroxy-LCB and/or Cer accumulation (Donahue et al., 2010; Ternes et al., 2011). Deciphering of Cer participation in the induction of HR and associated PCD also came from studies on accelerated cell death5 (acd5) and enhancing resistance to powdery mildew8 (RPW8)-mediated hypersensitive response (erh1) mutants, which displayed overaccumulation of Cers. These mutants exhibited spontaneous cell death and resistance to biotrophic pathogens, which seemed to be linked with SA and PR protein accumulation (Liang et al., 2003; Wang et al., 2008).Altogether, these data provide evidence of a link between PCD, defense, and sphingolipid metabolism. However, the fatty acid hydroxylase1/2 (atfah1/atfah2) double mutant that accumulates SA and Cers was more tolerant to the obligate biotrophic fungus Golovinomyces cichoracearum but did not display a PCD-like phenotype, suggesting that Cers alone are not involved in the induction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, but not trihydroxy-LCBs, might be primary mediators for LCB-induced PCD. The sphingoid base hydroxylase sbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD marker genes (Chen et al., 2008). On the contrary, increase in t18:0 was specifically sustained in plant interaction with the avirulent Pseudomonas syringae pv tomato (Pst) strain and correlated with a strong PCD induction in leaves (Peer et al., 2010). Thus, the nature of sphingolipids able to induce PCD is still under debate and may evolve depending on plants and their environment. The phosphorylated form of LCBs (LCB-Ps) could abrogate PCD induced by LCBs, Cers, or heat stress in a dose-dependent manner (Shi et al., 2007; Alden et al., 2011). Furthermore, blocking the conversion of LCBs to LCB-Ps by using specific inhibitors induced PCD in cell suspension culture (Alden et al., 2011). Recently, overexpression of rice (Oryza sativa) LCB kinase in transgenic tobacco (Nicotiana tabacum) plants reduced PCD after treatment with FB1 (Zhang et al., 2013). Genetic mutation on LCB-P lyase encoded by the AtDPL1 gene, modifying the LCB-LCB-P ratio, could impact PCD levels after treatment with FB1 (Tsegaye et al., 2007). Altogether, these data point to the existence of a rheostat between LCBs and their phosphorylated forms that controls plant cell fate toward cell death or survival.Data on plant sphingolipid functions are still fragmentary. Only a few reports have described interconnections between sphingolipids, cell death, and plant defense responses, almost exclusively in response to (hemi)biotrophic pathogens. Knowledge about such relations in response to necrotrophic pathogens is still in its infancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). In this report, the link between sphingolipids, cell death, and plant defense has been explored in response to Botrytis cinerea infection and in comparison with Pst infection. For this purpose, Atdpl1 mutant plants, disturbed in LCB/LCB-P accumulation without displaying any phenotype under standard growth conditions (Tsegaye et al., 2007), have been analyzed after pathogen infection. Our results revealed that modification of sphingolipid contents not only impacted plant tolerance to hemibiotrophs but also greatly affected resistance to necrotrophs. Whereas the SA signaling pathway is globally repressed in Atdpl1-1 compared with wild-type plants, the JA signaling pathway is significantly enhanced. Cell death and ROS accumulation are markedly modified in Atdpl1-1 mutant plants. We further demonstrated that phytosphingosine-1-phosphate (t18:0-P) and d18:0 are key players in pathogen-induced cell death and ROS generation. Here, we thus established a link between JA signaling, PCD, and sphingolipid metabolism.     

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.