Jean-Marie Guigonis

Effect of cadmium in the clam Ruditapes decussatus assessed by proteomic analysis.

Cadmium, an environmental stressor due to its toxicity, persistence and accumulation in biota, is widespread in the aquatic environment. Cadmium accumulation kinetics have revealed that Ruditapes decussatus has a high affinity to this metal. Proteomics is an effective tool to evaluate the toxic effects of contaminants. The aim of this study was to investigate the Cd effects in the gill and digestive gland of the sentinel species R. decussatus. Protein expression profiles (PEPs) in the clam tissues exposed to Cd (40 microg l(-1), 21 days) were compared to unexposed ones. Cd induces major changes in tissue-specific protein expression profiles in gill and digestive gland. This tissue dependent response results mainly from differences in Cd accumulation, protein inhibition and/or autophagy. An overall decrease of protein spots was detected in both treated tissues, being higher in gill. Some of the spots more drastically altered after pollutants exposure were excised and nine were identified by micro liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins identified by homology search in databases included: three proteins (8-fold) up-regulated, one down-regulated, four suppressed and one induced. Cd induces major changes in proteins involved in cytoskeletal structure maintenance (muscle-type actin, adductor muscle actin and beta-tubulin), cell maintenance (Rab GDP) and metabolism (ALDH and MCAD, both identified by de novo sequencing) suggesting potential energetic change. They provide a valuable knowledge of Cd effects at biochemical and molecular levels in the gill and digestive gland of R. decussatus.

Sulfenylated proteins in the Medicago truncatula–Sinorhizobium meliloti symbiosis☆

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)), play a crucial role as signaling molecules in the establishment and functioning of the nitrogen-fixing legume-Rhizobium symbiosis. The regulation of protein function through oxidative modification has emerged as an important molecular mechanism modulating various biological processes. Protein cysteine residues are known to be sensitive targets of H(2)O(2), in a posttranslational modification called sulfenylation. We trapped and identified sulfenylated proteins in the Medicago truncatula-Sinorhizobium meliloti symbiosis, by combining the use of chemical and genetic probes with mass spectrometry analysis. We identified 44 M. truncatula proteins sulfenylated in inoculated roots (two days post infection, 2dpi) and 65 such proteins in the functioning symbiotic organ, the nodule (four weeks post infection, 4wpi); 18 proteins were identified at both time points. However, the largest functional groups at 2dpi and 4wpi were different: redox state-linked proteins early in the interaction and proteins involved in amino-acid and carbohydrate metabolism in the nodule. Twenty proteins from S. meliloti, including some directly involved in nitrogen fixation, were also identified as sulfenylated. These results suggest that sulfenylation may regulate the activity of proteins playing major roles in the development and functioning of the symbiotic interaction.

Osmotically induced synthesis of the dipeptide N-acetylglutaminylglutamine amide is mediated by a new pathway conserved among bacteria

The dipeptide N-acetylglutaminylglutamine amide (NAGGN) was discovered in the bacterium Sinorhizobium meliloti grown at high osmolarity, and subsequently shown to be synthesized and accumulated by a few osmotically challenged bacteria. However, its biosynthetic pathway remained unknown. Recently, two genes, which putatively encode a glutamine amidotransferase and an acetyltransferase and are up-regulated by osmotic stress, were identified in Pseudomonas aeruginosa. In this work, a locus carrying the orthologous genes in S. meliloti, asnO and ngg, was identified, and the genetic and molecular characterization of the NAGGN biosynthetic pathway is reported. By using NMR experiments, it was found that strains inactivated in asnO and ngg were unable to produce the dipeptide. Such inability has a deleterious effect on S. meliloti growth at high osmolarity, demonstrating the key role of NAGGN biosynthesis in cell osmoprotection. β-Glucuronidase activity from transcriptional fusion revealed strong induction of asnO expression in cells grown in increased NaCl concentration, in good agreement with the NAGGN accumulation. The asnO–ngg cluster encodes a unique enzymatic machinery mediating nonribosomal peptide synthesis. This pathway first involves Ngg, a bifunctional enzyme that catalyzes the formation of the intermediate N-acetylglutaminylglutamine, and second AsnO, required for subsequent addition of an amide group and the conversion of N-acetylglutaminylglutamine into NAGGN. Interestingly, a strong conservation of the asnO–ngg cluster is observed in a large number of bacteria with different lifestyles, such as marine, symbiotic, and pathogenic bacteria, highlighting the ecological importance of NAGGN synthesis capability in osmoprotection and also potentially in bacteria host–cell interactions.

Additional bioactive guanidine alkaloids from the Mediterranean sponge Crambe crambe

The full chemical reinvestigation of the Mediterranean marine sponge Crambe crambe led to the isolation and structural characterization of 11 crambescin derivatives, including 8 new compounds, together with the known crambescidin 816. HRMS/MS studies allowed the complete assignment of the alkyl chain lengths of these guanidine alkaloids while the absolute configurations of all compounds were inferred from the comparison between experimental and theoretical circular dichroism spectra. Crambescidin 816 was proven to be more cytotoxic against neuronal cell lines than crambescin C1.

Heat Shock Cognate Protein 70 Regulates Gephyrin Clustering

Formation and stabilization of postsynaptic glycine receptor (GlyR) clusters result from their association with the polymerized scaffold protein gephyrin. At the cell surface, lateral diffusion and local trapping of GlyR by synaptic gephyrin clusters is one of the main factors controlling their number. However, the mechanisms regulating gephyrin/GlyR cluster sizes are not fully understood. To identify molecular binding partners able to control gephyrin cluster stability, we performed pull-down assays with full-length or truncated gephyrin forms incubated in a rat spinal cord extract, combined with mass spectrometric analysis. We found that heat shock cognate protein 70 (Hsc70), a constitutive member of the heat shock protein 70 (Hsp70) family, selectively binds to the gephyrin G-domain. Immunoelectron microscopy of mouse spinal cord sections showed that Hsc70 could be colocalized with gephyrin at inhibitory synapses. Furthermore, ternary Hsc70-gephyrin-GlyR coclusters were formed following transfection of COS-7 cells. Upon overexpression of Hsc70 in mouse spinal cord neurons, synaptic accumulation of gephyrin was significantly decreased, but GlyR amounts were unaffected. In the same way, Hsc70 inhibition increased gephyrin accumulation at inhibitory synapses without modifying GlyR clustering. Single particle tracking experiments revealed that the increase of gephyrin molecules reduced GlyR diffusion rates without altering GlyR residency at synapses. Our findings demonstrate that Hsc70 regulates gephyrin polymerization independently of its interaction with GlyR. Therefore, gephyrin polymerization and synaptic clustering of GlyR are uncoupled events.

Regulation of bud dormancy by manipulation of ABA in isolated buds of Rosa hybrida cultured in vitro

In vitro cultures showed that the proximal buds isolated from a rose (Rosa hybrida L. cv. Ruidriko Vivaldi®) stem were endodormant. Growth and a high percentage of bud break could be observed when cultures were treated with fluridone, an inhibitor of carotenoid synthesis. Flow cytometry determination of nuclear DNA content revealed that cell cycle activity of endodormant buds was arrested in the G 1 phase. Upon culture, the large decrease in bud ABA content was responsible for the progress from G1 to G2 phase whatever the culture medium. However, in control culture, neither cell division nor leaf primordium initiation could be observed and cells appeared stably arrested in G2 . By contrast, with fluridone, an additional ABA decrease was observed resulting from an inhibition of its synthesis inside the bud. New leaf primordia were initiated and many figures of mitosis could be observed, indicating that intense activity of cell division occurred after DNA replication. Therefore, the results indicate that, as long as ABA was synthesized inside the buds, cell cycle was arrested in G2 phase and buds remained dormant. Continued in situ ABA biosynthesis appears, therefore, to be required for the maintenance of bud dormancy.

Preventive Cancer Stem Cell‐Based Vaccination Reduces Liver Metastasis Development in a Rat Colon Carcinoma Syngeneic Model

Cancer stem cells (CSCs) represent a minor population of self‐renewing cancer cells that fuel tumor growth. As CSCs are generally spared by conventional treatments, this population is likely to be responsible for relapses that are observed in most cancers. In this work, we analyzed the preventive efficiency of a CSC‐based vaccine on the development of liver metastasis from colon cancer in a syngeneic rat model. We isolated a CSC‐enriched population from the rat PROb colon carcinoma cell line on the basis of the expression of the aldehyde dehydrogenase‐1 (ALDH1) marker. Comparative analysis of vaccines containing lysates of PROb or ALDHhigh cells by mass spectrometry identifies four proteins specifically expressed in the CSC subpopulation. The expression of two of them (heat shock protein 27‐kDa and aldose reductase) is already known to be associated with treatment resistance and poor prognosis in colon cancer. Preventive intraperitoneal administration of vaccines was then performed before the intrahepatic injection of PROb cancer cells. While no significant difference in tumor occurrence was observed between control and PROb‐vaccinated groups, 50% of the CSC‐based vaccinated animals became resistant to tumor development. In addition, CSC‐based vaccination induced a 99.5% reduction in tumor volume compared to the control group. To our knowledge, this study constitutes the first work analyzing the potential of a CSC‐based vaccination to prevent liver metastasis development. Our data demonstrate that a CSC‐based vaccine reduces efficiently both tumor volume and occurrence in a rat colon carcinoma syngeneic model. STEM CELLS2013;31:423–432

RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis†

The Tcirg1 gene encodes the osteoclast‐specific a3 isoform of the V‐ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP‐1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis.

Comparative Proteomics Study Reveals That Bacterial CpG Motifs Induce Tumor Cell Autophagy in Vitro and in Vivo

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.

Treatment of colon cancer cells using the cytosine deaminase/5-fluorocytosine suicide system induces apoptosis, modulation of the proteome, and Hsp90β phosphorylation

The bacterial cytosine deaminase (CD) gene, associated with the 5-fluorocytosine (5FC) prodrug, is one of the most widely used suicide systems in gene therapy. Introduction of the CD gene within a tumor induces, after 5FC treatment of the animal, a local production of 5-fluorouracil resulting in intratumor chemotherapy. Destruction of the gene-modified tumor is then followed by the triggering of an antitumor immune reaction resulting in the regression of distant wild-type metastasis. The global effects of 5FC on colorectal adenocarcinoma cells expressing the CD gene were analyzed using the proteomic method. Application of 5FC induced apoptosis and 19 proteins showed a significant change in 5FC-treated cells compared with control cells. The up-regulated and down-regulated proteins include cytoskeletal proteins, chaperones, and proteins involved in protein synthesis, the antioxidative network, and detoxification. Most of these proteins are involved in resistance to anticancer drugs and resistance to apoptosis. In addition, we show that the heat shock protein Hsp90β is phosphorylated on serine 254 upon 5FC treatment. Our results suggest that activation of Hsp90β by phosphorylation might contribute to tumor regression and tumor immunogenicity. Our findings bring new insights into the mechanism of the anticancer effects induced by CD/5FC treatment. [Mol Cancer Ther 2007;6(10):2747–56]