Reversible inactivation of the transcriptional function of P53 protein by farnesylation

BACKGROUND: The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function. RESULTS: We constructed an adenoviral vector coding a chimeric p53 protein by fusing p53 sequence with the 21 COOH term amino acids sequence of H-Ras. Indeed, the translation products of Ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a CAAX motif present at the C terminus of Ras proteins. The chimeric p53HRCaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. The farnesylated form of p53 resided mainly in the cytosol, where it is non-functional. Farnesyl transferase inhibitors (FTIs) specifically inhibited farnesyl isoprenoid lipid modification of proteins. Following treatment of the cells with an FTI, p53HRCaax underwent translocation into the nucleus where it retained transcription factor activity. Shifting p53 into the nucleus resulted in the induction of p21(waf1/CIP1 )and Bax transcription, cell growth arrest, caspase activation and apoptosis. CONCLUSION: Artificial protein farnesylation impaired the transcriptional activity of p53. This could be prevented by Farnesyl transferase inhibition. These data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria

A novel toxic effect of foodborne trichothecenes: The exacerbation of genotoxicity

International audienceTrichothecenes (TCT) are very common mycotoxins. While the effects of DON, the most prevalent TCT, have been extensively studied, less is known about the effect of other trichothecenes. DON has ribotoxic, pro-inflammatory, and cytotoxic potential and induces multiple toxic effects in humans and animals. Although DON is not genotoxic by itself, it has recently been shown that this toxin exacerbates the genotoxicity induced by model or bacterial genotoxins. Here, we show that five TCT, namely T-2 toxin (T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FX), and the newly discovered NX toxin, also exacerbate the DNA damage inflicted by various genotoxins. The exacerbation was dose dependent and observed with phleomycin, a model genotoxin, captan, a pesticide with genotoxic potential, and colibactin, a bacterial genotoxin produced by the intestinal microbiota. For this newly described effect, the trichothecenes ranked in the following order: T-2>DAS > FX > NIV ≥ DON ≥ NX. The genotoxic exacerbating effect of TCT correlated with their ribotoxic potential, as measured by the inhibition of protein synthesis. In conclusion, our data demonstrate that TCT, which are not genotoxic by themselves, exacerbate DNA damage induced by various genotoxins. Therefore, foodborne TCT could enhance the carcinogenic potential of genotoxins present in the diet or produced by intestinal bacteria

Oxygen concentration modulates colibactin production

International audienceUp to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue

MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling.

International audienceRho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules

Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity

International audienceOral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication

Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

International audienceIn Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4'-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies

Both EntD and ClbA can support the yersiniabactin siderophore synthesis <i>in vitro</i>.

<p>Siderophore production by the enterobactin and yersiniabactin siderophores producer <i>Escherichia coli</i> strain SE15 and derivatives. <b>A.</b> Chrome azurol S (CAS) plate upon which <i>E. coli</i> strain SE15 and derivatives have been streaked for overnight growth. <b>B.</b> Quantification of total siderophore production in supernatants of <i>E. coli</i> strain SE15 and derivatives determined by the CAS assay. The data are the means and standard deviations of 5 independent experiments. <b>C.</b> Quantification of the yersiniabactin siderophore production in <i>E. coli</i> strain SE15 and derivatives. <i>E. coli</i> strains HB101, MG1655 and DH5α were used as negative controls (K12). RLU: relative light units. ***: <i>P</i><0.001, **: <i>P</i><0.01, *: <i>P</i><0.05.</p

The presence of either EntD or ClbA is required to maintain full virulence of ExPEC.

<p>Mice underwent footpad injection with 10⁸ CFU of <i>E. coli</i> SP15 <i>entD clbA</i> strain and complemented derivatives. <b>A.</b> The percentage of mice survival was monitored. 10 to 25 mice were used per group. <b>B.</b> 18 h post infection 3 to 5 mice per group were sacrificed. Bacteria were quantified in spleen and blood collected from each animal. For statistical analysis, two-factor ANOVA and Bonferroni's multiple comparison test was performed. ***: <i>P</i><0.001, ns: not significant.</p