Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation

Rôle de la nucléoline et de macroH2A dans la structure et la fonction du nucléole

Ribosome synthesis starts in the nucleolus. The nucleolus structure is strongly influenced by ribosomal genes transcription. Ribosomal gene regulation is crucial for ribosome synthesis. The aim of this work was to define and open new methodological ways to characterize the role of macroH2A and nucleolin in the regulation of ribosomal genes expression. To date, MacroH2A is the only histone variant present in the nucleolus. Nucleolin is one of the major proteins of the nucleolus. The first part of this study is focused on the common carp, an original biological system, that adapts to temperature condition (winter and summer) by adjusting is gene expression. The results show that repression of ribosomal RNA expression, associated with a decrease of cellular activity, is concomitant with (i) nucleolar rearrangements, (ii) increase of macroH2A and nucleolin concentration and (iii) increase of DNA CpG methylation. Ribosomal genes expression appears to be linked to the level of macroH2A. To analyse the distribution of macroH2A on chromatin, we developed experiments based on extended chromatin fibers. We show that macroH2A colocalize with trimethylation of lysine 9 of H3, that is a marker of heterochromatin. Moreover, distribution of macroH2A is periodic, suggesting a role of this histone variant in a special chromatin organization. Finally, in the laboratory, we developed a cellular system based on nucleolin inhibition by RNA interference in HeLa cells. Two main results have emerged from the knockdown of nucleolin: decrease of pre-ribosomal RNA synthesis associated with nucleolar structure disruption and cell cycle arrest in mitosis leading to apoptosis. Thus, nucleolin is strongly implicated in the regulation of ribosomal genes expression.Le nucléole est le lieu où débute la synthèse des ribosomes. Sa structure est fortement corrélée à la transcription des gènes ribosomiques. La régulation de l'expression des gènes ribosomiques est une étape importante de la biogenèse des ribosomes. L'objectif de ce travail a été de définir et d'ouvrir des voies méthodologiques pour mettre en évidence le rôle de macroH2A et de la nucléoline dans la régulation de l'expression des gènes ribosomiques. MacroH2A est pour l'instant le seul variant d'histone présent dans le nucléole. La nucléoline est une des protéines majoritaires du nucléole. La première partie de notre étude a porté sur un système cellulaire original celui de la carpe dont l'adaptation aux conditions climatiques (hiver et été) requiert des changements importants dans l'expression génique. Ainsi la répression de l'expression des ARN ribosomiques, associée à une baisse d'activité cellulaire, est concomitante avec (i) une déstructuration du nucléole, (ii) une augmentation de la concentration de macroH2A et de la nucléoline et (iii) une augmentation de la méthylation d'îlots CpG. Il existe donc une corrélation entre l'expression des gènes ribosomiques et le niveau d'expression de macroH2A. Pour analyser la distribution de macroH2A sur une fibre de chromatine, nous avons utilisé la technique d'étirement de la chromatine. Nous avons montré que macroH2A co-localise avec la tri-méthylation de la lysine 9 de l'histone H3 qui est un marqueur de l'hétérochromatine. De plus, la distribution de macroH2A est périodique suggérant un rôle de cette histone dans une organisation particulière de la chromatine. Enfin, nous avons développé au laboratoire un système cellulaire reposant sur l'inhibition de la nucléoline par RNAi dans des cellules HeLa. Les conséquences de l'inhibition de la nucléoline sont multiples : une diminution de la synthèse de l'ARN pré-ribosomique accompagnée d'une perturbation de la structure du nucléole et d'un arrêt du cycle cellulaire en mitose pouvant conduire à l'apoptose. La nucléoline est donc largement impliquée dans la régulation de l'expression des gènes ribosomiques

Screening New Xylanase Biocatalysts from the Mangrove Soil Diversity

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia marina and Rhizophora stylosa trees during the wet and dry seasons and at two different sediment depths. GH11 xylanase diversity varied more according to tree species and season, than with respect to depth. One complete cDNA was selected (OFU29) and expressed in Pichia pastoris. The corresponding enzyme (called Xyn11-29) was biochemically characterized, revealing an optimal activity at 40–50 °C and at a pH of 5.5. Xyn11-29 was stable for 48 h at 35 °C, with a half-life of 1 h at 40 °C and in the pH range of 5.5–6. Xyn11-29 exhibited a high hydrolysis capacity on destarched wheat bran, with 40% and 16% of xylose and arabinose released after 24 h hydrolysis. Its activity on wheat straw was lower, with a release of 2.8% and 6.9% of xylose and arabinose, respectively. As the protein was isolated from mangrove sediments, the effect of sea salt on its activity was studied and discussed

Rôle de la nucléoline et de MacroH2A dans la structure et la fonction du nucléole

Le nucléole est le lieu où débute la synthèse des ribosomes. Sa structure est fortement corrélée à la transcription des gènes ribosomiques. La régulation de l expression des gènes ribosomiques est une étape importante de la biogenèse des ribosomes. L objectif de ce travail a été de définir et d ouvrir des voies méthodologiques pour mettre en évidence le rôle de macroH2A et de la nucléoline dans la régulation de l expression des gènes ribosomiques. MacroH2A est pour l instant le seul variant d histone présent dans le nucléole. La nucléoline est une des protéines majoritaires du nucléole. La première partie de notre étude a porté sur un système cellulaire original celui de la carpe dont l adaptation aux conditions climatiques (hiver et été) requiert des changements importants dans l expression génique. Ainsi la répression de l expression des ARN ribosomiques, associée à une baisse d activité cellulaire, est concomitante avec (i) une déstructuration du nucléole, (ii) une augmentation de la concentration de macroH2A et de la nucléoline et (iii) une augmentation de la méthylation d îlots CpG. Il existe donc une corrélation entre l expression des gènes ribosomiques et le niveau d expression de macroH2A. Pour analyser la distribution de macroH2A sur une fibre de chromatine, nous avons utilisé la technique d étirement de la chromatine. Nous avons montré que macroH2A co-localise avec la tri-méthylation de la lysine 9 de l histone H3 qui est un marqueur de l hétérochromatine. De plus, la distribution de macroH2A est périodique suggérant un rôle de cette histone dans une organisation particulière de la chromatine. Enfin, nous avons développé au laboratoire un système cellulaire reposant sur l inhibition de la nucléoline par RNAi dans des cellules HeLa. Les conséquences de l inhibition de la nucléoline sont multiples : une diminution de la synthèse de l ARN pré-ribosomique accompagnée d une perturbation de la structure du nucléole et d un arrêt du cycle cellulaire en mitose pouvant conduire à l apoptose. La nucléoline est donc largement impliquée dans la régulation de l expression des gènes ribosomiques.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication.

BACKGROUND: Nucleolin is a major component of the nucleolus, but is also found in other cell compartments. This protein is involved in various aspects of ribosome biogenesis from transcription regulation to the assembly of pre-ribosomal particles; however, many reports suggest that it could also play an important role in non nucleolar functions. To explore nucleolin function in cell proliferation and cell cycle regulation we used siRNA to down regulate the expression of nucleolin. RESULTS: We found that, in addition to the expected effects on pre-ribosomal RNA accumulation and nucleolar structure, the absence of nucleolin results in a cell growth arrest, accumulation in G2, and an increase of apoptosis. Numerous nuclear alterations, including the presence of micronuclei, multiple nuclei or large nuclei are also observed. In addition, a large number of mitotic cells showed a defect in the control of centrosome duplication, as indicated by the presence of more than 2 centrosomes per cell associated with a multipolar spindle structure in the absence of nucleolin. This phenotype is very similar to that obtained with the inactivation of another nucleolar protein, B23. CONCLUSION: Our findings uncovered a new role for nucleolin in cell division, and highlight the importance of nucleolar proteins for centrosome duplication

An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters

International audienc

CHARAKTERIZATION OF THE HYBRID PROLINE-RICH PROTEIN FAMILY IN POTATO AND FUNCTIONAL ANALYSIS OF THE StHyPRP1 GENE

<p><b>Copyright information:</b></p><p>Taken from "Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication"</p><p>http://www.biomedcentral.com/1471-2199/8/66</p><p>BMC Molecular Biology 2007;8():66-66.</p><p>Published online 10 Aug 2007</p><p>PMCID:PMC1976620.</p><p></p>d siRNA against nucleolin (mix siRNA #2 and #4) treated cells. At the indicated times, HeLa and human primary fibroblast cells were harvested and protein extracts were analyzed by Western blotting with anti-nucleolin antibody. Equal loading was verified using anti-tubulin antibodies. The amount of B23 protein was also analyzed using anti-B23 antibodies. B. Quantification of the Western blots. Data were normalized to the tubulin protein. The normalized nucleolin protein level was set to 100% in control cells. Data are from three independent experiments. C. Quantitative RT-PCR. At indicated times after transfection, HeLa and human primary fibroblast cells were harvested, total RNA was isolated and used for cDNA synthesis and quantitative PCR with nucleolin or cytoplasmic β-actin specific primers. Data were normalized to the amount of β-actin mRNA. Untransfected control and scrambled (transfected with scrambled siRNA) cells were also used and all data were normalized to the amount of nucleolin mRNA in control cells. Data are from three independent experiments. D. Immunofluorescence analysis. Four days after siRNA transfection (control scrambled siRNA #1, panels A, or mix of siRNA #2 and #4 panels B), HeLa cells were examined by immunofluorescence using anti-nucleolin antibody in green. DNA was counterstained with DAPI (Blue). In panels A.1 to A.3 and B.1 to B.3, a zoom on one representative cell is shown to highlight the modification of the shape and number of nucleolar structures after nucleolin depletion. Bars represent 10 μm

Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication-2

<p><b>Copyright information:</b></p><p>Taken from "Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication"</p><p>http://www.biomedcentral.com/1471-2199/8/66</p><p>BMC Molecular Biology 2007;8():66-66.</p><p>Published online 10 Aug 2007</p><p>PMCID:PMC1976620.</p><p></p>ells were counted at the indicated time after transfection. The graph represents the average of 5 independent experiments. B. Analysis of cell cycle in nucleolin depleted cells. Untransfected and transfected HeLa cells were stained with propidium iodide and processed for FACS analysis. Histograms from a representative experiment showing G1 and G2/M populations shaded in red and S phase population in blue. Sub G1 represent apoptotic cell population. The vertical and the horizontal axes represent respectively cell number and DNA fluorescence intensity. The percentages of cells in individual cell cycle phases were quantified with ModFit LD software. C. Mitotic index of HeLa cells and human primary fibroblasts transfected with control siRNA or the siRNA mix #2 and #4. After 4 days of transfection, cell populations were stained with an anti phospho S-H3 antibody and cyclin B1 to detect mitotic cells. Positive cells were scored by microscopic observation. This graph represents the average of the different experiments (DAPI, PH3S28 and cyclin B1) shown in additional file

Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication-0

<p><b>Copyright information:</b></p><p>Taken from "Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication"</p><p>http://www.biomedcentral.com/1471-2199/8/66</p><p>BMC Molecular Biology 2007;8():66-66.</p><p>Published online 10 Aug 2007</p><p>PMCID:PMC1976620.</p><p></p>d siRNA against nucleolin (mix siRNA #2 and #4) treated cells. At the indicated times, HeLa and human primary fibroblast cells were harvested and protein extracts were analyzed by Western blotting with anti-nucleolin antibody. Equal loading was verified using anti-tubulin antibodies. The amount of B23 protein was also analyzed using anti-B23 antibodies. B. Quantification of the Western blots. Data were normalized to the tubulin protein. The normalized nucleolin protein level was set to 100% in control cells. Data are from three independent experiments. C. Quantitative RT-PCR. At indicated times after transfection, HeLa and human primary fibroblast cells were harvested, total RNA was isolated and used for cDNA synthesis and quantitative PCR with nucleolin or cytoplasmic β-actin specific primers. Data were normalized to the amount of β-actin mRNA. Untransfected control and scrambled (transfected with scrambled siRNA) cells were also used and all data were normalized to the amount of nucleolin mRNA in control cells. Data are from three independent experiments. D. Immunofluorescence analysis. Four days after siRNA transfection (control scrambled siRNA #1, panels A, or mix of siRNA #2 and #4 panels B), HeLa cells were examined by immunofluorescence using anti-nucleolin antibody in green. DNA was counterstained with DAPI (Blue). In panels A.1 to A.3 and B.1 to B.3, a zoom on one representative cell is shown to highlight the modification of the shape and number of nucleolar structures after nucleolin depletion. Bars represent 10 μm

Seasonal environmental changes regulate the expression of the histone variant macroH2A in an eurythermal fish.

International audienc