Burkholderia cenocepacia causes Cepacia syndrome, a fatal pneumonia that affects Cystic Fibrosis patients. Aromatic degradation has been linked to virulence in B. cenocepacia by insertional mutagenesis of genes involved in phenylacetic acid catabolism. B. cenocepacia has two paralogous copies of PaaK, the phenylacetyl-CoA ligase, which produces PA-CoA, the inducer of the pathway. Our objective was to assess a role for PaaK1 and PaaK2 in PA metabolism and virulence by constructing clean deletion mutants for each gene, and a double paaK mutant, as well as to quantify virulence using the nematode host model. Deletion and complementation of paaK1 revealed no change in killing phenotype. Reporter activity assays revealed PaaK1-dependent induction of the PA pathway 3-hydroxyphenylacetic acid but not 4-hydroxyphenylacetic acid. Altogether, these results demonstrate that 3-OHPA induces the PA degradation pathway in a paaK1 dependent manner and that PaaK1 is not involved in pathogenicity.

Burkholderia cenocepacia causes Cepacia syndrome, a fatal pneumonia that affects Cystic Fibrosis patients. Aromatic degradation has been linked to virulence in B. cenocepacia by insertional mutagenesis of genes involved in phenylacetic acid catabolism. B. cenocepacia has two paralogous copies of PaaK, the phenylacetyl-CoA ligase, which produces PA-CoA, the inducer of the pathway. Our objective was to assess a role for PaaK1 and PaaK2 in PA metabolism and virulence by constructing clean deletion mutants for each gene, and a double paaK mutant, as well as to quantify virulence using the nematode host model. Deletion and complementation of paaK1 revealed no change in killing phenotype. Reporter activity assays revealed PaaK1-dependent induction of the PA pathway 3-hydroxyphenylacetic acid but not 4-hydroxyphenylacetic acid. Altogether, these results demonstrate that 3-OHPA induces the PA degradation pathway in a paaK1 dependent manner and that PaaK1 is not involved in pathogenicity.

Stuttgart, Universiẗat, Diss., 2007.

Köln, Universiẗat, Diss., 2003.

Bibliography: pages 181-201. / A genomic library of the collagenolytic Vibrio alginolyticus strain was established in Escherichia coli HB101 employing the positive selection vector pEcoR251. A glutamine synthetase (GS) gene, glnA was identified by complementation of the glnA deletion in E. coli ET8051 glnA, glnL, glnG deletion strain. The glnA region of V. alginolyticus was cloned on a 5.7 kb insert in pRM210.

L’incidence du mélanome a augmenté de façon considérable lors des trente dernières années avec un doublement tous les dix ans. Le mélanome ne représente que 5% des cancers cutanés mais entraîne 80% de décès, ce qui constitue un problème majeur de santé publique. En effet, cette tumeur est extrêmement agressive et possède un fort potentiel métastatique. Dès l’apparition de métastases, le pronostic vital devient fortement défavorable. Malgré des avancées thérapeutiques majeures, de nombreux patients sont encore réfractaires à ces nouveaux traitements. La compréhension des mécanismes impliqués dans le développement de cette tumeur reste donc un enjeu de premier ordre. Le séquençage d'exomes a conduit à l'identification d'une mutation dans le gène RAC1 (la mutation P29S) constituant une des mutations somatiques les plus fréquentes dans le mélanome (après les mutations BRAFV600, NRASQ61 et NF1). RAC1 est une petite GTPase qui fonctionne dans plusieurs processus cellulaires. Dans des conditions physiologiques, l'activité de RAC1 est principalement contrôlée par des protéines activatrices de l'activité GTPase (GAPs) et des facteurs d'échange Nucléotidique (GEF). GAPs et GEFs contrôlent le niveau de RAC1-GTP et régulent donc son activité. L'activité de RAC1 est aussi dépendante de son niveau d'expression protéique qui est contrôlé par des E3 ubiquitine ligases, parmi lesquelles HACE1. HACE1 est considérée comme un suppresseur de tumeur. De façon inattendue, les données obtenues montrent clairement que HACE1 favorise les propriétés migratoires et tumorigéniques des cellules de mélanome. / Melanoma incidence has considerably increased over the last thirty years, with a doubling every ten years. Melanoma accounts for only 5% of cutaneous cancers but causes more than 80% of deaths, which is a major public health problem. Indeed, this tumor is extremely aggressive and has a high metastatic potential. After the onset of metastases, the prognosis becomes highly unfavorable. Despite major therapeutic advances, many patients are still refractory to these new treatments. Understanding the mechanisms involved in the development of this tumor and the identification of new therapies remain a major issue. The sequencing of exomes led to the identification of a mutation in the RAC1 gene (P29S) constituting one of the most frequent somatic mutations in melanoma (after the BRAFV600, NRASQ61 and NF1 mutations). RAC1 is a small GTPase that is involved in several key cellular processes. Under physiological conditions, the activity of RAC1 is mainly controlled by GTPase activating proteins (GAPs) and Nucleotide Exchange (GEF) exchange factors. GAPs and GEFs control the level of RAC1- GTP and thus regulate its activity. The activity of RAC1 is also dependent on its protein level of expression which is controlled by E3 ubiquitin ligases, including HACE1. HACE1 is considered a tumor suppressor. Unexpectedly, our data clearly show that HACE1 promotes migratory and tumorigenic properties of melanoma cells. Indeed, inhibition of HACE1 alters migration of melanoma cells in vitro, as well as in vivo pulmonary colonization in mice. Transcriptomic analysis of 4 melanoma cell lines demonstrated that HACE1 suppression inhibits ITGAV and ITGB1 expression.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

No description available.

The significance of poxvirus manipulation of the host ubiquitin proteasome system has become increasingly apparent. Ubiquitin is post-translationally added to target proteins by a highly conserved enzymatic cascade, typically resulting in protein degradation via the 26S proteasome. The highly conserved poxvirus protein, p28, is a functional ubiquitin ligase and a critical virulence factor. Here, we investigate the relationship between p28 and ubiquitination. We observed that the KilA-N DNA binding domain in p28 targeted p28 to viral factories, where p28 co-localized with conjugated ubiquitin. Furthermore, we determined that p28 is highly regulated by ubiquitination and proteasomal degradation. Disruption of p28 ubiquitin ligase activity revealed that p28 is regulated through auto-ubiquitination and ubiquitination by an additional unknown ubiquitin ligase. Moreover, we observed Lysine-48 ubiquitin linkages, Lysine-63 ubiquitin linkages and a proteasomal subunit co-localizing with p28 at the viral factory, suggesting an intricate relationship between p28 and proteasomal degradation. / Virology