Cortactin overexpression results in sustained epidermal growth factor receptor signaling by preventing ligand-induced receptor degradation in human carcinoma cells

The chromosome 11q13 region is frequently amplified in human carcinomas and results in an increased expression of various genes including cortactin, and is also associated with an increased invasive potential. Cortactin acts as an important regulator of the actin cytoskeleton. It is therefore very tempting to speculate that cortactin is the crucial gene within the 11q13 amplicon that mediates the invasive potential of these carcinomas. Cortactin also participates in receptor-mediated endocytosis, and recent findings have shown that, during receptor internalization, cortactin overexpression inhibits the ubiquitylation-mediated degradation of the epidermal growth factor receptor, resulting in a sustained ligand-induced epidermal growth factor receptor activity

Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes

Identification of Surprisingly Diverse Type IV Pili, across a Broad Range of Gram-Positive Bacteria

In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes.We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins

Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its Nor C-terminal end

International audienceLinker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter

Ready, set, internalize: mechanisms and regulation of GLUT4 endocytosis

The facilitative glucose transporter GLUT4, a recycling membrane protein, is required for dietary glucose uptake into muscle and fat cells. GLUT4 is also responsible for the increased glucose uptake by myofibres during muscle contraction. Defects in GLUT4 membrane traffic contribute to loss of insulin-stimulated glucose uptake in insulin resistance and Type 2 diabetes. Numerous studies have analysed the intracellular membrane compartments occupied by GLUT4 and the mechanisms by which insulin regulates GLUT4 exocytosis. However, until recently, GLUT4 internalization was less well understood. In the present paper, we review: (i) evidence supporting the co-existence of clathrin-dependent and independent GLUT4 internalization in adipocytes and muscle cells; (ii) the contrasting regulation of GLUT4 internalization by insulin in these cells; and (iii) evidence suggesting regulation of GLUT4 endocytosis in muscle cells by signals associated with muscle contraction

YopH prevents monocyte chemoattractant protein 1 expression in macrophages and T-cell proliferation through inactivation of the phosphatidylinositol 3-kinase pathway

Phosphatidylinositol 3-kinase (PI 3-kinase) and its target protein kinase B (Akt) are involved in various processes including internalization, chemotaxis and proliferation. We analysed the activation of Akt in J774 macrophages infected with virulent (pYV+) or avirulent (pYV-) Yersinia enterocolitica. During the early stage of infection with pYV+ and pYV- bacteria, Akt and its targets, glycogen synthase kinase 3 (GSK-3) and forkhead transcription factor (FKHRL1), became phosphorylated. This phosphorylation induction was inhibited by wortmannin and thus dependent on PI 3-kinase. When infection was carried out with pYV+ bacteria but not with pYV- bacteria, Akt and its targets became dephosphorylated at later time points. Using single knock-out mutants in bacterial effector genes, we have determined that the tyrosine phosphatase YopH was responsible for the inactivation of the PI 3-kinase cascade. In macrophages, this inactivation correlated with the downregulation of mRNA coding for monocyte chemoattractant protein 1 (MCP-1), suggesting that YopH inhibits recruitment of macrophages to lymph nodes. We also analysed the effects of Y. enterocolitica infection on the proliferation of T lymphocytes. Consistent with the observation that YopH inactivated the Akt pathway, YopH inhibited PI 3-kinase-dependent secretion of interleukin 2 and proliferation. These data reveal a new effect of YopH in Yersinia pathogenesis

Regulation of mRNA expression in macrophages after Yersinia enterocolitica infection : role of different Yop effectors

The Yop virulon, which comprises a complete type III secretion system and secreted proteins, allows bacteria from the genus Yersinia to resist the nonspecific immune response of the host. This virulon, which is encoded by a plasmid called pYV in Yersinia enterocolitica, enables extracellular bacteria to inject six Yop effectors (YopE, -H, -T, -O, -P, -M) into the host cell. To investigate the role of YopP, YopM, and the other pYV-encoded factors on the expression of the host cell genes, we characterized the transcriptome alterations in infected mouse macrophages using the microarray technique. PU5-1.8 macrophages were infected either with an avirulent (pYV(-)), a wild type (pYV(+)), or two knockout (yopP(-) and yopM(-)) mutants of Y. enterocolitica. Expression alterations in response to Y. enterocolitica infection were monitored for 6657 genes. Among those, 857 genes were affected, 339 of which were specifically regulated by the action of the Yop virulon. Further analysis of those 339 genes allowed identification of specific targets of YopP, YopM, or the other pYV-encoded factors. According to these results, the main action of the Yop virulon is to counteract the host cell pro-inflammatory response to the infection. YopP participates to this inhibition, whereas another pYV-encoded factor appears to also be involved in this down-regulation. Besides, YopM was found to induce the regulation of genes involved in cell cycle and cell growth, revealing for the first time an in vitro effect for YopM. In addition to YopM, other pYV factors distinct from YopP affected the expression of genes involved in cycling. In conclusion, these results provide new insight into the mechanisms of Yersinia pathogenicity by identifying the changes in host genes expression after infection and highlight the concerted actions of the different Yop effectors

Revisiting absorption and electronic circular dichroism spectra of cholesterol in solution: A joint experimental and theoretical study

International audienceCholesterol is doubtless one of the most studied bio-molecules, which unfortunately features low emitting properties, precluding its in vivo study by fluorescence experiments. The design of fluorescent analogues of cholesterol is thus an appealing challenge in biochemistry, which simultaneously requires minor changes in its chemical structure (to retain main biological properties) and considerable enhancement of light emission. To this aim, the photochemical behaviour of the native molecule has to be deeply understood. In this work, we focused our attention on the electronic absorption of cholesterol in several common organic solutions, combining experimental (through ultraviolet-visible and electronic circular dichroism spectroscopy) and theoretical approaches (at the time-dependent density functional theory level) in order to solve the important discrepancies previously reported in the literature on the maximum absorption wavelengths and on the nature (Rydberg and/or π → π*) of the associated electronic transition