Septin 9_i2 is downregulated in tumors, impairs cancer cell migration and alters subnuclear actin filaments

International audienceFunctions of septin cytoskeletal polymers in tumorigenesis are still poorly defined. Their role in the regulation of cytokinesis and cell migration were proposed to contribute to cancer associated aneuploidy and metastasis. Overexpression of Septin 9 (Sept9) promotes migration of cancer cell lines. SEPT9 mRNA and protein expression is increased in breast tumors compared to normal and peritumoral tissues and amplification of SEPT9 gene was positively correlated with breast tumor progression. However, the existence of multiple isoforms of Sept9 is a confounding factor in the analysis of Sept9 functions. In the present study, we analyze the protein expression of Sept9_i2, an uncharacterized isoform, in breast cancer cell lines and tumors and describe its specific impact on cancer cell migration and Sept9 cytoskeletal distribution. Collectively, our results showed that, contrary to Sept9_i1, Sept9_ i2 did not support cancer cell migration, and induced a loss of subnuclear actin filaments. These effects were dependent on Sept9_i2 specific N-terminal sequence. Sept9_i2 was strongly down-regulated in breast tumors compared to normal mammary tissues. Thus our data indicate that Sept9_i2 is a negative regulator of breast tumorigenesis. We propose that Sept9 tumorigenic properties depend on the balance between Sept9_i1 and Sept9_i2 expression levels

Kinetic analysis of mouse brain proteome alterations following chikungunya virus infection before and after appearance of clinical symptoms

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV in

Cooperation between Engulfment Receptors: The Case of ABCA1 and MEGF10

The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway

The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells

Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach

<p>Abstract</p> <p>Background</p> <p>Over its life cycle, the <it>Plasmodium falciparum </it>parasite is exposed to different environmental conditions, particularly to variations in O₂pressure. For example, the parasite circulates in human venous blood at 5% O₂pressure and in arterial blood, particularly in the lungs, at 13% O₂pressure. Moreover, the parasite is exposed to 21% O₂levels in the salivary glands of mosquitoes.</p> <p>Methods</p> <p>To study the metabolic adaptation of <it>P. falciparum </it>to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken.</p> <p>Results</p> <p>Even though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response.</p> <p>Conclusions</p> <p>These results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of <it>P. falciparum</it>. This study provides a better understanding of the adaptive capabilities of <it>P. falciparum </it>to environmental changes and may lead to the development of novel therapeutic targets.</p

The lack of NF-kappa B transactivation and PKC epsilon expression in CD4(+)CD8(+) thymocytes correlates with negative selection.

Deletion of autoreactive thymocytes at the DP stage is the basis for tolerance to thymus-expressed self antigens. In this study we investigated whether distinct signalling pathways are induced in DP thymocytes as compared to mature T cells upon stimulation with antigen. Using triple transgenic mice expressing a TCR transgene, dominant negative ras/Mek proteins and a reporter gene construct with AP-1 or NF-kappa B binding sites, we showed a complete lack of transcriptional activity of NF-kappa B but not AP-1 in DP thymocytes, whereas both were transcriptionally active in mature T cells after antigenic stimulation. Lack of NF-kappa B induction correlated with increased death in response to antigen. AP-1 induction was dependent on the integrity of the ras/Mek pathway indicating that this pathway was activated in the DP thymocytes. In contrast, we found a complete lack of constitutive expression of the epsilon isoform of Protein Kinase C (PKC) in DP thymocytes, although it was present in mature thymocytes and peripheral T cells. Taken together the results suggest that the lack of PKC epsilon in DP thymocytes could lead to the absence of NF-kappa B activity after antigenic stimulation contributing to negative selection. Cell Death and Differentiation (2000) 7, 1253 - 1262

Proteomic analysis revealed alterations of the Plasmodium falciparum metabolism following salicylhydroxamic acid exposure

Marylin Torrentino-Madamet1, Lionel Almeras2, Christelle Travaill&amp;eacute;1, V&amp;eacute;ronique Sinou1, Matthieu Pophillat3, Maya Belghazi4, Patrick Fourquet3, Yves Jammes5, Daniel Parzy11UMR-MD3, Universit&amp;eacute; de la M&amp;eacute;diterran&amp;eacute;e, Antenne IRBA de Marseille (IMTSSA, Le Pharo), 2Unit&amp;eacute; de Recherche en Biologie et Epid&amp;eacute;miologie Parasitaires, Antenne IRBA de Marseille (IMTSSA, Le Pharo), 3Centre d&amp;#39;Immunologie de Marseille Luminy, Institut National de la Sant&amp;eacute; et de la Recherche M&amp;eacute;dicale, Centre National de la Recherche Scientifique, Universit&amp;eacute; de la M&amp;eacute;diterran&amp;eacute;e, 4Centre d&amp;#39;Analyse Prot&amp;eacute;omique de Marseille, Institut F&amp;eacute;d&amp;eacute;ratif de Recherche Jean Roche, Facult&amp;eacute; de M&amp;eacute;decine Nord, 5UMR-MD2, Physiologie et Physiopathologie en Conditions d&amp;#39;Oxyg&amp;eacute;nations Extr&amp;ecirc;mes, Institut F&amp;eacute;d&amp;eacute;ratif de Recherche Jean Roche, Facult&amp;eacute; de M&amp;eacute;decine Nord, Marseille, FranceObjectives: Although human respiratory metabolism is characterized by the mitochondrial electron transport chain, some organisms present a &amp;ldquo;branched respiratory chain.&amp;rdquo; This branched pathway includes both a classical and an alternative respiratory chain. The latter involves an alternative oxidase. Though the Plasmodium falciparum alternative oxidase is not yet identified, a specific inhibitor of this enzyme, salicylhydroxamic acid (SHAM), showed a drug effect on P. falciparum respiratory function using oxygen consumption measurements. The present study aimed to highlight the metabolic pathways that are affected in P. falciparum following SHAM exposure.Design: A proteomic approach was used to analyze the P. falciparum proteome and determine the metabolic pathways altered following SHAM treatment. To evaluate the SHAM effect on parasite growth, the phenotypic alterations of P. falciparum after SHAM or/and hyperoxia exposure were observed.Results: After SHAM exposure, 26 proteins were significantly deregulated using a fluorescent two dimensional-differential gel electrophoresis. Among these deregulated proteins, some were particularly involved in energetic metabolism. And the combinatory effect of SHAM/hyperoxia seems deleterious for the growth of P. falciparum.Conclusion: Our results indicated that SHAM appears to activate glycolysis and decrease stress defense systems. These data provide a better understanding of parasite biology.Keywords: Plasmodium falciparum, salicylhydroxamic acid, hyperoxia, glycolysis, proteomi

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC = 0.06 mg.L-1) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route

Western blot validations of differentially regulated proteins identified by 2D-DIGE and/or iTRAQ analyses.

<p>(A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock (M), early (E), late paralytic (LP) and late tetanus-like (LT), separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye; green) and the specific immunoreactive proteins (FITC or Cy5 dye; red). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean ± S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons. The significance of the differential protein expression are indicated *, p<0.05; **, p<0.01; ***, p<0.001. A.U., arbitrary units. ANXA2: annexin A2; ARRB1: β-arrestin; GABRA1: γ-aminobutyric acid receptor subunit alpha-1; GRASP1: GRIP-associated protein; ITGAV: integrin αV; MYPT1: myosin phosphatase target subunit 1; N-Ras: N-Ras; RABEP1: rabaptin-5; SYNGR3: synaptogyrin-3.</p