A simple SDS-Page protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae)

International audiencePremise of the study - Carnivorous plants have always fascinated scientists because these plants are able to attract, capture and digest animal prey using their remarkable traps that contain digestive secretions. Nepenthes is one of the largest genera of carnivorous plants, with 120 species described thus far. Despite an outstanding diversity of trap designs, many species are often confused with each other and remain difficult to classify because they resemble pitchers or of the occurrence of interspecific hybrids. Methods - Here, we propose a new method to easily distinguish Nepenthes species based on a 1D SDS PAGE protein pattern analysis of their pitcher secretions. Intraspecific comparisons were performed between specimens growing in different environmental conditions to ascertain the robustness of this method. Key results - Our results show that, at the juvenile stage and in the absence of prey in the pitcher, an examined species is characterized by a specific and stable profile, whatever the environmental conditions. Conclusions - The method we describe here can be used as a reliable tool to easily distinguish between Nepenthes species and to help with potential identification based on the species-specific protein pattern of their pitcher secretions, which is complementary to the monograph informatio

Alterations in Cellular Energy Metabolism Associated with the Antiproliferative Effects of the ATM Inhibitor KU-55933 and with Metformin

<div><p>KU-55933 is a specific inhibitor of the kinase activity of the protein encoded by <em>Ataxia telangiectasia mutated</em> (ATM), an important tumor suppressor gene with key roles in DNA repair. Unexpectedly for an inhibitor of a tumor suppressor gene, KU-55933 reduces proliferation. In view of prior preliminary evidence suggesting defective mitochondrial function in cells of patients with Ataxia Telangiectasia (AT), we examined energy metabolism of cells treated with KU-55933. The compound increased AMPK activation, glucose uptake and lactate production while reducing mitochondrial membrane potential and coupled respiration. The stimulation of glycolysis by KU-55933 did not fully compensate for the reduction in mitochondrial functions, leading to decreased cellular ATP levels and energy stress. These actions are similar to those previously described for the biguanide metformin, a partial inhibitor of respiratory complex I. Both compounds decreased mitochondrial coupled respiration and reduced cellular concentrations of fumarate, malate, citrate, and alpha-ketogluterate. Succinate levels were increased by KU-55933 levels and decreased by metformin, indicating that the effects of ATM inhibition and metformin are not identical. These observations suggest a role for ATM in mitochondrial function and show that both KU-55933 and metformin perturb the TCA cycle as well as oxidative phosphorylation.</p> </div

Proteome analysis of digestive fluids in Nepenthes pitchers

International audienceBackground and Aims: Carnivorous plants have developed strategies to enable growth in nutrient-poor soils. For the genus Nepenthes, this strategy represents producing pitcher-modified leaves that can trap and digest various prey. These pitchers produce a digestive fluid composed of proteins, including hydrolytic enzymes. The focus of this study was on the identification of these proteins.Methods: In order to better characterize and have an overview of these proteins, digestive fluid was sampled from pitchers at different stages of maturity from five species of Nepenthes (N. mirabilis, N. alata, N. sanguinea, N. bicalcarata and N. albomarginata) that vary in their ecological niches and grew under different conditions. Three complementary approaches based on transcriptomic resources, mass spectrometry and in silico analysis were used.Key Results: This study permitted the identification of 29 proteins excreted in the pitchers. Twenty of these proteins were never reported in Nepenthes previously and included serine carboxypeptidases, α- and β-galactosidases, lipid transfer proteins and esterases/lipases. These 20 proteins display sequence signals allowing their secretion into the pitcher fluid.Conclusions:Nepenthes pitcher plants have evolved an arsenal of enzymes to digest prey caught in their traps. The panel of new proteins identified in this study provides new insights into the digestive process of these carnivorous plants

Effects of KU-55933 and metformin on TCA metabolites.

<p>(<b>A</b>) TCA metabolites were measured by NMR. <i>P</i> values for changes in TCA metabolites are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s003" target="_blank">Table S1</a> (<b>B</b>) Interpretation of metabolic changes observed. <i>[a]</i> ATM is hypothesized to have a role in oxidative phosphorylation, effecting respiratory complex II. Therefore, the ATM inhibitor KU-55933 leads not only to reduced ATP production, but also to accumulation of succinate. <i>[b]</i> KU-55933 also may reduce oxidative phosphorylation by a mechanism involving SCO2, as discussed in the text. <i>[c]</i> Metformin acts to inhibit oxidative phosphorylation, but prior evidence together with our findings of decreased NAD⁺ suggest a site of action involving respiratory complex I. <i>[d]</i> Both KU-55933 and metformin exposure lead to increased glucose uptake and lactate production, consistent with a compensatory increase in glycolysis following decreased oxidative phosphorylation. <i>[e]</i> Our observations provide evidence for reduced concentrations of TCA cycle intermediates with exposure to either KU-55933 or metformin, but we postulate different reasons for this: metformin may reduce TCA cycle activity because of a reduction in supply of complex I-generated NAD⁺, while KU-55933 may act to inhibit conversion of succinate to fumarate. ATM, Ataxia Telangiectasia Mutated protein; SCO2, Synthesis of Cytochrome C Oxidase 2; AMPK, AMP-activated protein kinase; TSC1/TSC2, Tuberous Sclerosis 1/Tuberous Sclerosis 2; mTOR, Mammalian Target of Rapamycin complex 1; rpS6, ribosomal protein S6.</p

Inhibition of ATM by KU-55933 decreases SCO2 expression in MCF-7 cells.

<p>MCF-7 cells were exposed to KU-55933 (10 µM) for the indicated time. After harvesting, cells were lysed and prepared for immunoblot analyses using antibodies against SCO2, phospho-ATM (Ser¹⁹⁸¹), ATM, phosphorylated p53 (Ser¹⁵), p53, phospho-S6 (Ser^235/236), S6rp, phospho-AMPK (Thr¹⁷²) and AMPK. ß-actin is shown as a loading control. The results are representative of three individual experiments.</p

Effects of KU-55933 and metformin on metabolism in MCF-7 cells.

<p>Cells were exposed to KU (10 µM) or metformin (5 mM) for 72 hrs. (<b>A</b>) The effect of KU-55933 or metformin on viable cell number was measured by counting cells able to exclude Trypan blue. Cell number was significantly reduced by KU-55933 (*<i>P</i> = 0.0042) and by metformin (**<i>P</i> = 0.0011). (<b>B</b>) Lactate production was significantly increased in cells treated with KU-55933 (*<i>P</i> = 0.0218) or metformin (**<i>P</i> = 0.0012). (<b>C</b>) Glucose consumption was increased with exposure to either KU-55933 (*<i>P</i> = 0.0463) or metformin (**<i>P</i> = 0.0058) treated cells. (<b>D</b>) Both KU-55933 and metformin decreased ATP levels in MCF-7 cells. Results are the mean ± S.E (<i>n</i> = 4). (KU-55933 compared to control *<i>P</i> = 0.0015 and metformin compared to control ** <i>P</i> = 0.0005). (<b>E</b>) Cells were incubated with JC-1 (2 µM), or H₂O₂ (100 µM, used to activate ATM by oxidative stress), or rotenone (1 µM), or FCCP (1 µM). Mitochondrial membrane potential was probed with JC-1 and visualized by flow cytometry. Loss of mitochondrial membrane potential (ΔΨ) is indicated by a decrease in FL2/FL1 fluorescence intensity ratio <b>(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s001" target="_blank">Figure S1</a> for flow cytometry data set)</b>. Results are expressed as mean ± S.E.M. (<i>n</i> = 4). KU-55933 (*<i>P</i> = 0.0003) and metformin (** <i>P</i><0.0001) both significantly decreased ΔΨ. (<b>F</b>) Total cellular respiration (black bars, left y-axis) of MCF-7 cells treated with KU-55933 or metformin was compared with untreated cells. Results are the mean ± S.E.M. (KU-55933 compared to control *<i>P</i> = 0.0045, and metformin compared to control ** <i>P</i> = 0.0496). Uncoupled respiration was determined in the presence of oligomycin. The percentage of uncoupled respiration was calculated as: (uncoupled respiration/total mitochondrial respiration), and is shown by hatched bars, right y-axis. (<b>G</b>) KU-55933 or metformin treatment increased cell death (<b>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s002" target="_blank">Figure S2</a> for flow cytometry data set)</b>. Bars represent percentage of necrotic cells. Results are expressed as the mean ± S.E.M. (<i>n</i> = 3) in duplicate (KU-55933 compared to control *<i>P</i> = 0.0005, and metformin compared to control **<i>P</i> = 0.0299). (<b>H</b>) KU-55933 or metformin treatment resulted in increased apoptosis <b>(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s002" target="_blank">Figure S2</a> for flow cytometry data set).</b> Bars represent percentage of apoptotic cells. Results are expressed as the mean ± S.E.M. (n = 3) in duplicate (KU-55933 compared to control *<i>P</i><0.0001, and metformin compared to control **<i>P</i> = 0.0458).</p

Subcellular localization of ATM.

<p>Total MCF-7 cell lysate and MCF-7 cells fractionated into cytoplasmic, nuclear and mitochondrial extracts were immunoblotted with ATM antibody, α-Tubulin (cytoplasmic marker), Ki67 (nuclear marker) and VDAC (mitochondrial marker). The results indicate ATM immunoreactivity in mitochondrial extracts that are negative for cytoplasmic and nuclear markers.</p

Growth inhibition by the ATM inhibitor KU-55933 and metformin.

<p>(<b>A</b>) MCF-7 (LKB^+/+) and (<b>B</b>) HeLa (LKB^−/−) cancer cells in exponential stages of growth were seeded into 96-well plates with 10% FBS and after 24 hrs exposed to increasing concentrations of KU-55933 (ATM inhibitor) in media containing 1% FBS for 72 hrs. Cell growth was estimated by Alamar Blue dye reduction (resazurin (3 µM)). Data are presented as mean ± S.E.M. from 3 independent experiments done in triplicate. (<b>C–F</b>) MCF-7 HepG2, HeLa and MCF-10A cells were growth inhibited by KU-55933 and metformin. Cells were seeded into 96-well plates in the presence of 1% FBS and after 24 hrs treated with KU-55933 (10 µM) or metformin (5 mM). Data are presented as mean ± S.E.M. from 4 independent experiments done in triplicate. * indicates a result significantly different from that obtained in the absence of KU-55933 or metformin as determined by 2-way ANOVA (<i>P</i><0.0001). (<b>G</b>) MCF-7 cells were transfected with 50 nM ATM-siRNA or with control siRNA. Twenty-four hours after transfection, cells were treated with KU-55933 (10 µM) or metformin (5 mM) and incubated for 48 hrs in RPMI containing 1% FBS. Cell growth in each well was measured by counting cells using Trypan blue. Results using cell number or Alamar blue as endpoints yielded the same conclusions. Columns, mean of 3 independent experiments carried out in triplicate (n = 9); bars, S.E.M. (<b>H</b>) After transfecting MCF-7 cells with 50 nM ATM-siRNA or with control siRNA, cells were lysed and prepared for immunoblot analyses using antibodies against ATM. ß-actin is shown as a loading control.</p