Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3-and viral-induced expression of type i interferons

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression. © 2012 Nature America, Inc. All rights reserved.

Pellino3 targets RIP1 and regulates the pro-apoptotic effects of TNF-∝

Tumour necrosis factor-a (TNF) can activate NF-?B to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-?B pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

Hydrological and photochemical influence on dynamics of estuarine bacteria

Os estuários são ecossistemas complexos, onde os processos físicos, químicos e biológicos estão intimamente ligados. A dinâmica bacteriana num estuário reflete a interação e a elevada variação temporal e espacial desses processos. Este trabalho teve como objetivo elucidar as interações entre os processos físicos, fotoquímicos e microbiológicos no sistema estuarino da Ria de Aveiro (Portugal). Para tal, foi realizada uma abordagem inicial no campo, durante a qual as comunidades bacterianas na coluna de água foram caracterizadas em termos de abundância e atividade ao longo de 2 anos. O estudo foi realizado em dois locais distintos, escolhidos por tipificarem as características marinhas e salobras do estuário. Estes locais possuem diferentes hidrodinâmicas, influências fluviais e, quantidade e composição de matéria orgânica. Numa perspectiva mecanicista, foram realizadas simulações laboratoriais no sentido de elucidar a resposta das bactérias à matéria orgânica foto-transformada. As comunidades bacterianas no estuário adaptam-se a diferentes regimes de água doce, desenvolvendo padrões de abundância e atividade distintos nas zonas marinha e salobra. Os elevados caudais dos rios induzem estratificação vertical na zona marinha, promovendo o fluxo de fitoplâncton do mar para o estuário, do bacterioplâncton do estuário para o mar, e estimulam a importação de bactérias aderentes a partículas na zona salobra. O transporte advectivo e os processos de ressuspensão contribuem para aumentar 3 vezes o número de bactérias aderentes a partículas durante os períodos de intensas descargas fluviais. Adicionalmente, a atividade bacteriana no estuário é controlada pela concentração de azoto inerente à variações de água doce. O fornecimento de azoto em associação com a fonte dos substratos bacterianos induzem alterações significativas na produtividade. O padrão de variação vertical de comunidades bacterianas foi distinto nas duas zonas do estuário. Na zona marinha, as bactérias na microcamada superficial (SML) apresentaram taxas de hidrólise mais elevadas, mas menores taxas de incorporação de monómeros e produção de biomassa que na água subjacente (UW), enquanto na zona salobra, as taxas de hidrólise e incorporação foram similares nos dois compartimentos, mas a produtividade foi significativamente mais elevada na SML. Apesar da abundância bacteriana ter sido semelhante na SML e UW, a fração de células aderentes a partículas foi significativamente maior na SML (2-3 vezes), em ambas as zonas do estuário. A integração dos resultados microbiológicos com as variáveis ambientais e hidrológicos mostraram que fortes correntes na zona marinha promovem a mistura vertical, inibindo o estabelecimento de uma comunidade bacteriana na SML distinta da UW. Em contraste, na zona de água salobra, a menor velocidades das correntes fornece as condições adequadas ao aumento da atividade bacteriana na SML. Características específicas do local, tais como a hidrodinâmica e as fontes e composição da matéria orgânica, conduzem também a diferentes graus de enriquecimento superficial de matéria orgânica e inorgânica, influenciando a sua transformação. Em geral, o ambiente da SML estuarina favorece a hidrólise de polímeros, mas inibe a utilização de monómeros, comparativamente com água subjacente. No entanto, as diferenças entre as duas comunidades tendem a atenuar-se com o aumento da atividade heterotrófica na zona salobra. A matéria orgânica dissolvida cromófora (CDOM) das duas zonas do estuário possui diferentes características espectrais, com maior aromaticidade e peso molecular médio (HMW) na zona de água salobra, em comparação com a zona marinha. Nesta zona, a abundância bacteriana correlacionou-se com a350 e a254, sugerindo uma contribuição indireta das bactéria para HMW CDOM. A irradiação do DOM resultou numa diminuição dos valores de a254 e a350, e, em um aumento do declive S275-295 e dos rácios E2:E3 (a250/a365) e SR. No entanto, a extensão de transformações foto-induzidas e as respostas microbianas são dependentes das características iniciais CDOM, inferidas a partir das suas propriedades ópticas. A dinâmica estuarina influencia claramente as atividades heterotróficas e a distribuição dos microorganismos na coluna de água. A entrada de água doce influencia a dinâmica e os principais reguladores das comunidades bacterianas no estuário. Os processos fotoquímicos e microbianos produzem alterações nas propriedades ópticas da CDOM e a combinação desses processos determina o resultado global e o destino da CDOM nos sistemas estuarinos com influência na produtividade nas áreas costeiras adjacente.

Dissection of the early steps in the assembly of the 20S proteasome in Saccharomyces cerevisiae

Tese de dout., Bioquímica (Biologia Celular e Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Hydrogen peroxide sensing, signaling and regulation of transcription factors

The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and, (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M-1s−1 and ≥ 1.3 × 103 M-1s−1 were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

The role of the TRAF-interacting protein in proliferation and differentiation.

Ubiquitination of proteins is a post-translational modification, which decides on the cellular fate of the protein. Addition of ubiquitin moieties to proteins is carried out by the sequential action of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3, ubiquitin ligase. The TRAF-interacting protein (TRAIP, TRIP, RNF206) functions as Really Interesting New Gene (RING)-type E3 ubiquitin ligase, but its physiological substrates are not yet known. TRAIP was reported to interact with TRAF [tumor necrosis factor (TNF) receptor-associated factors] and the two tumor suppressors CYLD and Syk (spleen tyrosine kinase). Ectopically expressed TRAIP was shown to inhibit nuclear factor-kappa B (NF-κB) signalling. However, recent results suggested a role for TRAIP in biological processes other than NF-κB regulation. Knock-down of TRAIP in human epidermal keratinocytes repressed cellular proliferation and induced a block in the G1/S phase of the cell cycle without affecting NF-κB signalling. TRAIP is necessary for embryonal development as mutations affecting the Drosophila homologue of TRAIP are maternal effect-lethal mutants, and TRAIP knock-out mice die in utero because of aberrant regulation of cell proliferation and apoptosis. These findings underline the tight link between TRAIP and cell proliferation. In this review, we summarize the data on TRAIP and put them into a larger perspective regarding the role of TRAIP in the control of tissue homeostasis.

Molecular mechanisms for the regulation of skin homeostasis

L'ubiquitination est une modification des protéines conservée, consistant en l'addition de résidus « ubiquitine » et régulant le destin cellulaire des protéines. La protéine « TRAF-interacting protein » TRAIP (ou TRIP) est une ligase E3 qui catalyse l'étape finale de l'ubiquitination. TRAIP est conservé dans l'évolution et est nécessaire au développement des organismes puisque l'ablation de TRAIP conduit à la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la réduction de l'expression de TRAIP dans des kératinocytes épidermiques humains réprime la prolifération cellulaire et induit un arrêt du cycle cellulaire en phase Gl, soulignant le lien étroit entre TRAIP et la prolifération cellulaire. Comme les mécanismes de régulation de la prolifération jouent un rôle majeur dans l'homéostasie de la peau, il est important de caractériser la fonction de TRAIP dans ces mécanismes. En utilisant des approches in vitro, nous avons déterminé que la protéine TRAIP est instable, modifiée par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont également révélé que l'expression de TRAIP est dépendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifié le facteur de transcription E2F1 comme en étant le responsable, en régulant directement le promoteur de TRAIP. Aussi, TRAIP endogène ou surexprimée est surtout localisée au niveau du nucléole, une organelle nucléaire qui est désassemblée pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imagé la protéine TRAIP fusionnée à une protéine fluorescente, à l'intérieur de cellules vivantes nommées HeLa, à l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localisée autour des chromosomes en début de mitose, puis est arrangée au niveau de l'ADN chromosomique en fin de mitose. La détection de TRAIP endogène à l'aide d'un anticorps spécifique a confirmé cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interférence ARN a inhibé leur capacité à s'arrêter en milieu de mitose. Nos résultats suggèrent que le mécanisme sous-jacent peut être lié au point de contrôle de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knock¬out is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

[Portrait de garçon en uniforme de collégien à mi-jambes, debout portant un brassard de communiant] : [photographie] / Pierre Eugène Fixon

Ancien possesseur : Gilles, Albert (1873-1959)

[Jeune garçon au papillon, à mi-jambes, assis, de trois-quarts à gauche] : [photographie] / Non identifié

Ancien possesseur : Gilles, Albert (1873-1959)

[Portrait de jeune garçon, à mi-genoux, debout, de trois-quarts à gauche, l'avant-bras droit reposant sur une balustrade] : [photographie] / Derussy

Ancien possesseur : Gilles, Albert (1873-1959)

[Portrait de fillette, à mi-corps, debout, de face, un cerceau à la main] : [photographie] / Jean-Baptiste Sabatier-Blot

Ancien possesseur : Gilles, Albert (1873-1959)

Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.