Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Hypertension increases connexin43 in a tissue-specific manner.

BACKGROUND: Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. METHODS AND RESULTS: Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart. CONCLUSIONS: The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

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In the last years, the classical view of glial cells (in particular of astrocytes) as a simple supportive cell for neurons has been replaced by a new vision in which glial cells are active elements of the brain. Such a new vision is based on the existence of a bidirectional communication between astrocytes and neurons at synaptic level. Indeed, perisynaptic processes of astrocytes express active G-protein-coupled receptors that are able (1) to sense neurotransmitters released from the synapse during synaptic activity, (2) to increase cytosolic levels of calcium, and (3) to stimulate the release of gliotransmitters that in turn can interact with the synaptic elements. The mechanism(s) by which astrocytes can release gliotransmitter has been extensively studied during the last years. Many evidences have suggested that a fraction of astrocytes in situ release neuroactive substances both with calcium-dependent and calcium-independent mechanism(s); whether these mechanisms coexist and under what physiological or pathological conditions they occur, it remains unclear. However, the calcium-dependent exocytotic vesicular release has received considerable attention due to its potential to occur under physiological conditions via a finely regulated way. By releasing gliotransmitters in millisecond time scale with a specific vesicular apparatus, astrocytes can integrate and process synaptic information and control or modulate synaptic transmission and plasticity.

The protease activity of the paracaspase MALT1 is controlled by monoubiquitination.

The protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how this activity is controlled remains unknown. Here we identify a monoubiquitination of MALT1 on Lys644 that activated the protease function of MALT1. Monoubiquitinated MALT1 had enhanced protease activity, whereas a ubiquitination-deficient MALT1 mutant with replacement of that lysine with arginine (MALT1(K644R)) had less protease activity, which correlated with impaired induction of interleukin 2 (IL-2) via the T cell antigen receptor in activated T cells. Expression of MALT1(K644R) diminished the survival of cells derived from diffuse large B cell lymphoma of the activated B cell-like subtype (ABC DLBCL), which require constitutive protease activity of MALT1 for survival. Thus, monoubiquitination of MALT1 is essential for its catalytic activation and is therefore a potential target for the treatment of ABC-DLBCL and for immunomodulation.

Colon-specific deletion of epithelial sodium channel causes sodium loss and aldosterone resistance.

Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.

A tumor-specific cellular environment at the brain invasion border of adamantinomatous craniopharyngiomas.

Craniopharyngiomas (CP) are benign epithelial tumors of the sellar region and can be clinicopathologically distinguished into adamantinomatous (adaCP) and papillary (papCP) variants. Both subtypes are classified according to the World Health Organization grade I, but their irregular digitate brain infiltration makes any complete surgical resection difficult to obtain. Herein, we characterized the cellular interface between the tumor and the surrounding brain tissue in 48 CP (41 adaCP and seven papCP) compared to non-neuroepithelial tumors, i.e., 12 cavernous hemangiomas, 10 meningiomas, and 14 metastases using antibodies directed against glial fibrillary acid protein (GFAP), vimentin, nestin, microtubule-associated protein 2 (MAP2) splice variants, and tenascin-C. We identified a specific cell population characterized by the coexpression of nestin, MAP2, and GFAP within the invasion niche of the adamantinomatous subtype. This was especially prominent along the finger-like protrusions. A similar population of presumably astroglial precursors was not visible in other lesions under study, which characterize them as distinct histopathological feature of adaCP. Furthermore, the outer tumor cell layer of adaCP showed a distinct expression of MAP2, a novel finding helpful in the differential diagnosis of epithelial tumors in the sellar region. Our data support the hypothesis that adaCP, unlike other non-neuroepithelial tumors of the central nervous system, create a tumor-specific cellular environment at the tumor-brain junction. Whether this facilitates the characteristic infiltrative growth pattern or is the consequence of an activated Wnt signaling pathway, detectable in 90% of these tumors, will need further consideration.

Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication

Rapport de synthèse : L'infection par le virus de l'hépatite C (VHC) a une évolution sévère chez les patients co-infectés par VIH/VHC, de même que chez les patients transplantés hépatiques. Toutefois, les mécanismes impliqués dans cette évolution restent peu clairs. Dans ce travail, nous étudions le profil fonctionnel des cellules T spécifiques dirigées contre le virus de l'hépatite C chez 86 patients mono-infectés par VHC, 48 patients co-infectés par VIH/VHC et 42 patients ayant bénéficié d'une transplantation hépatique. La production d'IFN-Y et d'IL-2 et la capacité de proliférer des cellules T CD4+ et CD8+ sont évaluées après stimulation par des peptides dérivés du VHC. Chez les patients mono-infectés par le VHC, les cellules T spécifiques au VHC sont polyfonctionnelles du point de vue de la sécrétion de cytokines, avec trois profils de sécrétion pour les cellules T CD4+: sécrétion uniquement de IL-2, sécrétion de IL-2 et IFN-y et sécrétion uniquement de IFN-gamma, et de deux profils pour les cellules T CD8+: sécrétion de IL-2 et IFN-y et sécrétion uniquement de IFN-gamma. En revanche, les cellules T spécifiques au VHC chez les individus coinfectés par VIH/VHC et chez les patients transplantés hépatiques ont un profil de sécrétions de cytokines marqué par l'absence de cellules CD4+ sécrétant uniquement l'Il-2 et l'absence de cellules CD8+ sécrétant à la fois IL-2 et IFN-gamma. De plus, la prolifération de cellules T CD4+ et CD8+ spécifiques au VHC est considérablement réduite chez les patients co-infectés par VIH/VHC, comme chez les transplantés hépatiques. La présence de cellules T effectrices uniquement (définies par l'absence de cellules T CD4+ sécrétants uniquement de l'IL-2 et l'absence de cellules T CD8+ sécrétant à la fois IL-2 et IFN-gamma et altération de la capacité proliférative) est associée avec une charge virale VHC significativement plus élevée et une fibrose hépatique plus sévère. Par conséquent, les présents résultats suggèrent la participation de mécanismes immunitaires dans l'évolution accélérée de l'hépatite C chez les patients co-infectés par VIH-1 et chez les patients greffés hépatiques.

GENETIC AND BIOCHEMICAL ANALYSIS OF THE TESTIS-SPECIFIC ZINC FINGER PROTEIN CTCFL/BORIS

Abstract en FrançaisCTCFL a d'abord été identifié comme un paralogue de la protéine ubiquitaire CTCF en raison de sa forte homologie entre leurs onze « zinc fingers », un domaine de liaison à l'ADN. Parmi ses nombreux rôles, la liaison des zinc fingers de CTCF à la région de contrôle de l'empreinte (ICR) maternelle non-méthylée Igf2/H19, contrôle l'expression empreinte (monoallélique) de H19 et IGF2 dans les cellules somatiques. La méthylation de l'ICR Igf2/H19 paternelle est nécessaire à l'expression empreinte de ces deux gènes. Bien que le mécanisme par lequel l'ICR est méthylé soit mal compris, il est connu que l'établissement de la méthylation se produit pendant le développement des cellules germinales mâles et que les ADN méthyltransférases de novo DNMT3A et DNMT3L sont essentiels. Par conséquent, CTCFL fournit un bon candidat pour un rôle dans la méthylation de l'ICR paternelle Igf2/H19 en raison de son expression restreinte à certains types de cellules où la méthylation de l'ICR a lieu (spermatogonies et spermatocytes) ainsi qu'en raison sa capacité à lier les ICR lgf2/HÎ9 dans ces cellules. Les premiers travaux expérimentaux de cette thèse portent sur le rôle possible des mutations de CTCFL chez les patients atteints du syndrome de Silver-Russell (SRS), où une diminution de la méthylation de l'ICR IGF2/H19 a été observée chez 60% d'entre eux. Admettant que CTCFL pourrait être muté chez ces patients, j'ai examiné les mutations possibles de CTCFL chez 35 d'entre eux par séquençage de l'ADN et analyse du nombre de copies d'exons. N'ayant trouvé aucune mutation chez ces patients, cela suggère que les mutations de CTCFL ne sont pas associées au SRS. Les travaux expérimentaux suivants ont porté sur les modifications post-traductionnelles de CTCFL par la protéine SU MO « small ubiquitin-like modifier » (SUMO). La modification de protéines par SU MO change les interactions avec d'autres molécules (ADN ou protéines). Comme CTCFL régule sans doute l'expression d'un certain nombre de gènes dans le cancer et que plusieurs facteurs de transcription sont régulés par SUMO, j'ai mené des expériences pour déterminer si CTCFL est sumoylé. En effet, j'ai observé que CTCFL est sumoylated in vitro et in vivo et j'ai déterminé les deux résidus d'attachement de SUMO aux lysines 181 et 645. Utilisant les mutants de CTCFL K181R et K645R ne pouvant pas être sumoylated, j'ai évalué les conséquences fonctionnelles de la modification par SUMO. Je n'ai trouvé aucun changement significatif dans la localisation subcellulaire, la demi-vie ou la liaison à l'ADN, mais ai constaté que la sumoylation module à la fois {'activation CTCFL-dépendante et la répression de l'expression génique. Il s'agit de la première modification post-traductionnelle décrite pour CTCFL et les conséquences possibles de cette modification sont discutées pour le cancer et les testicules normaux. Avec cette thèse, j'espère avoir ajouté des résultats importants à l'étude de CTCFL et donné quelques idées pour de futures recherches.AbstractJeremiah Bernier-Latmani, Institute of Pathology, University of Lausanne, CHUVCTCFL was first identified as a paralog of the ubiquitous protein CTCF because of high homology between their respective eleven zinc fingers, a DNA binding domain. Among its many roles, CTCF zinc finger-mediated binding to the unmethylated maternal Igf2/H19 imprinting control region (ICR), controls the imprinted (monoallelic) expression of Igf2 and H19 in somatic cells. Methylation of the paternal Igf2/H19 ICR is necessary for the imprinted expression of the two genes. Although the mechanism by which the ICR is methylated is incompletely understood, it is known that establishment of methylation occurs during male germ cell development and the de novo DNA methyltransferases DNMT3A and DNMT3L are essential. Therefore, CTCFL provided a good candidate to play a role in methylation of the paternal Igf2/H19 ICR because of its restricted expression to cell types where ICR methylation takes place (spermatogonia and spermatocytes) and its ability to bind the Igf2/H19 ICR in these cells. The first experimental work of this thesis investigated the possible role of CTCFL mutations in Silver-Russell syndrome (SRS) patients, where it has been observed that 60% of the patients have reduced methylation of the IGF2/HÎ9 ICR. Reasoning that CTCFL could be mutated in these patients, I screened 35 patients for mutations in CTCFL by DNA sequencing and exon copy number analysis, I did not find any mutations in these patients suggesting that mutations of CTCFL are not associated with SRS. The next experimental work of my thesis focused on posttranslational modification of CTCFL by small ubiquitin-like modifier (SUMO) protein. SUMO modification of proteins changes the interactions with other molecules (DNA or protein). As CTCFL arguably regulates the expression of a number of genes in cancer and many transcription factors are regulated by SUMO, I conducted experiments to assess whether CTCFL is sumoylated. I found that CTCFL is sumoylated in vitro and in vivo and determined the two residues of SUMO attachment to be lysines 181 and 645. Using K181R, K645R mutated CTCFL- which cannot be detected to be sumoylated-1 assessed the functional consequences of SUMO modification. I found no significant changes in subcellular localization, half-life or DNA binding, but found that sumoylation modulates both CTCFL-dependent activation and repression of gene expression. This is the first posttranslational modification described for CTCFL and possible consequences of this modification are discussed in both cancer and normal testis. With this thesis, I hope I have added important findings to the study of CTCFL and provide some ideas for future research.

Three-dimensional patient-specific dosimetry in radioimmunotherapy with 90Y-ibritumomab-tiuxetan.

Aim of the present article was to perform three-dimensional (3D) single photon emission tomography-based dosimetry in radioimmunotherapy (RIT) with (90)Y-ibritumomab-tiuxetan. A custom MATLAB-based code was used to elaborate 3D images and to compare average 3D doses to lesions and to organs at risk (OARs) with those obtained with planar (2D) dosimetry. Our 3D dosimetry procedure was validated through preliminary phantom studies using a body phantom consisting of a lung insert and six spheres with various sizes. In phantom study, the accuracy of dose determination of our imaging protocol decreased when the object volume decreased below 5 mL, approximately. The poorest results were obtained for the 2.58 mL and 1.30 mL spheres where the dose error evaluated on corrected images with regard to the theoretical dose value was -12.97% and -18.69%, respectively. Our 3D dosimetry protocol was subsequently applied on four patients before RIT with (90)Y-ibritumomab-tiuxetan for a total of 5 lesions and 4 OARs (2 livers, 2 spleens). In patient study, without the implementation of volume recovery technique, tumor absorbed doses calculated with the voxel-based approach were systematically lower than those calculated with the planar protocol, with average underestimation of -39% (range from -13.1% to -62.7%). After volume recovery, dose differences reduce significantly, with average deviation of -14.2% (range from -38.7.4% to +3.4%, 1 overestimation, 4 underestimations). Organ dosimetry in one case overestimated, in the other underestimated the dose delivered to liver and spleen. However, both for 2D and 3D approach, absorbed doses to organs per unit administered activity are comparable with most recent literature findings.

Inducible kidney-specific Sgk1 knockout mice show a salt-losing phenotype.

The expression of the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by mineralocorticoids and, in turn, upregulates the renal epithelial Na(+) channel (ENaC). Total inactivation of Sgk1 has been associated with transient urinary Na(+) wasting with a low-Na(+) diet, while the aldosterone-mediated ENaC channel activation was unchanged in the collecting duct. Since Sgk1 is ubiquitously expressed, we aimed to study the role of renal Sgk1 and generated an inducible kidney-specific knockout (KO) mouse. We took advantage of the previously described TetOn/CreLoxP system, in which rtTA is under the control of the Pax8 promotor, allowing inducible inactivation of the floxed Sgk1 allele in the renal tubules (Sgk1fl/fl/Pax8/LC1 mice). We found that under a standard Na(+) diet, renal water and Na(+)/K(+) excretion had a tendency to be higher in doxycycline-treated Sgk1 KO mice compared with control mice. The impaired ability of Sgk1 KO mice to retain Na(+) increased significantly with a low-salt diet despite higher plasma aldosterone levels. On a low-Na(+) diet, the Sgk1 KO mice were also hyperkaliuric and lost body weight. This phenotype was accompanied by a decrease in systolic and diastolic blood pressure. At the protein level, we observed a reduction in phosphorylation of the ubiquitin protein-ligase Nedd4-2 and a decrease in the expression of the Na(+)-Cl(-)-cotransporter (NCC) and to a lesser extent of ENaC.

Epithelial to mesenchymal transition in the pathogenesis of uterine malignant mixed Müllerian tumours: the role of ubiquitin proteasome system and therapeutic opportunities.

Malignant mixed Müllerian tumours (malignant mixed mesodermal tumours, MMMT) of the uterus are metaplastic carcinomas with a sarcomatous component and thus they are also called carcinosarcomas. It has now been accepted that the sarcomatous component is derived from epithelial elements that have undergone metaplasia. The process that produces this metaplasia is epithelial to mesenchymal transition (EMT), which has recently been described as a neoplasia-associated programme shared with embryonic development and enabling neoplastic cells to move and metastasise. The ubiquitin proteasome system (UPS) regulates the turnover and functions of hundreds of cellular proteins. It plays important roles in EMT by being involved in the regulation of several pathways participating in the execution of this metastasis-associated programme. In this review the specifi c role of UPS in EMT of MMMT is discussed and therapeutic opportunities from UPS manipulations are proposed.

Ubiquitination and the Ubiquitin-Proteasome System as regulators of transcription and transcription factorsin epithelial mesenchymal transition of cancer.

Epithelial to Mesenchymal Transition (EMT) in cancer is a process that allows cancer cells to detach from neighboring cells, become mobile and metastasize and shares many signaling pathways with development. Several molecular mechanisms which regulate oncogenic properties in neoplastic cells such as proliferation, resistance to apoptosis and angiogenesis through transcription factors or other mediators are also regulators of EMT. These pathways and downstream transcription factors are, in their turn, regulated by ubiquitination and the Ubiquitin-Proteasome System (UPS). Ubiquitination, the covalent link of the small 76-amino acid protein ubiquitin to target proteins, serves as a signal for protein degradation by the proteasome or for other outcomes such as endocytosis, degradation by the lysosome or directing these proteins to specific cellular compartments. This review discusses aspects of the regulation of EMT by ubiquitination and the UPS and underlines its complexity focusing on transcription and transcription factors regulating EMT and are being regulated by ubiquitination.

Lats2, a novel regulator of Elongin BC ubiquitin ligase

Résumé : Le Large tumor suppressor, Lats2, est une protéine humaine homologue au suppresseur de tumeur Warts (Lats) de Drosophila melanogaster, qui réprime la prolifération des cellules en altérant leur cycle au niveau des transitions Gl/S et G2/M, et en induisant l'apoptose. Pourtant, la voie moléculaire par laquelle Lats2, une sériase-thréonine kinase, déclenche l'arrêt du cycle cellulaire, est toujours inconnue. Notre équipe a d'abord déterminé que Lats2 était un gène de réponse à la protéine p53 (Kostic et al., 2000). Par la suite, nous avons identifié des protéines interagissant avec Lats2, notamment les modules de reconnaissance du substrat des ligases Colline E3 (des protéines contenant Socs box ou F box) ainsi que deux Bous-unités du Signalosome CSN: CSN4 et CSNS. En outre, Lats2 est connue pour s'associer au Super-complexe composé de CSN et des ligases Colline E3 (Rongere, thesis, 2004; Rongere, unpublished results, 2005). Le travail présenté ici sur Lats2 a confirmé que cette protéine est une kinase associée à CSN. Nous avons caractérisé les interactions spécifiques de domaines de Lats2 avec hSocs3, hWsb 1 (des protéines Socs box) et hFBX-7 (une protéine F box), ainsi que les conséquences physiologiques des interactions avec hSocs3, hWsb1 et hSocs1. Des expériences de GST pull-down ont montré que les deux domaines, N-terminal et kinase, de Lats2 interagissent avec hSocs3, hWsb1 et hFBX-7, ce qui suggère aussi que l'ensemble de la protéine Lats2 est impliqué dans ces interactions. Une étude approfondie des interactions entre Lats2 et hSocs3 indique que le domaine kinase de Lats2 interagit avec la région de hSocs3 contenant un domaine SH2, situé en amont du domaine Socs box de hSocs3. Par ailleurs, Lats2 phosphoryle des régions spécifiques entre les domaines N-terminal et SH2 (Sl), et, entre les domaines SH2 et Socs box (S3) de la protéine hSocs3. Ces résultats révèlent que hSocs3 est un.nouveau substrat de Lats2. Des modifications de l'activité kinase ont aussi révélé que la protéine sauvage Lats2 (wt Lats2) était capable de phosphoryler hSocs3, alors qu'un mutant dead du domaine kinase Lats (poche ATP délétée, Lats2OATP) non. L'analyse des mutations a permis d'identifier deux résidus sériase situés aux positions 1441145 (S3), spécifiquement phosphorylés par wt Lats2. La phosphorylation des protéines représentant un signal de dégradation protéolytique, nous avons envisagé que Lats2 pouvait cibler hSocs3 pour une dégradation protéasomale. Lorsque wt Lats2 est surexprimée dans des cellules HEK293T et COS7, la demi-vie de hSocs3, un élément de la ligase Elongine BC-Colline É3 (ligase EBC), diminue significativement, effet que n'a pas la surexpression de Lats2OATP. De plus, la stabilité de hSocs3 dépend de la phosphorylation des résidus sériase aux positions 144/145 par wt Lats2. Bien que les sites de phosphorylation ne soient pas définis pour les deux autres modules de reconnaissance du substrat de la ligase EBC: hWsb 1 et hSocsl, leurs demi-vies diminuent également quand wt Lats2 est surexprimée. Pour les tests in vivo, nous avons synthétisé des esiRNA pour diminuer l'expression du gène endogène lats2, ce qui a entraîné une augmentation d'un facteur 2 de la demi-vie de hSocs3 et de hWsbl dans les cellules HEK293T. En conclusion, nos résultats suggérent que Lats2, une kinase associée au CSN, est un nouveau régulateur de la fonction des ligases EBC, agissant sur le renouvellement des protéines hSocs3, hSocs1 et hWsb1. Ainsi, Lats2 altère la spécificité et la capacité des ligases EBC, régulant par là même la stabilité de nombreuses protéines, ciblées par les ligases EBC pour une dégradation protéasomale. D'autres études devraient révéler si la modification observée de la fonction de la ligase EBC par Lats2, associée au Super-complexe, est également responsable du renouvellement des régulateurs du cycle cellulaire et des changements dans ce même cycle observés lors de la surexpression de Lats2. Summary : The Large tumor suppressor 2 (Lats2) is a human homologue of the Drosophila melanogaster tumor suppressor Warts (Cats) who negatively regulates cell proliferation by altering cell cycle Gl/S and G2/M transition and inducing apoptosis. However, the molecular pathway by which Lats2, a serine-threonine kinase, mediates cell cycle arrest is still unknown. Lats2 was initially identified to be a p53 response gene by our group (Kostic et al., 2000). Subsequently, our group identified interacting candidates of Lats2, including substrate recognition modules of Cullin-based E3 ligases (Socs box or F-box containing proteins) as well as two subunits of the Signalosome (CSN), CSN4 and CSNS. Additionally, Lats2 was shown to associate with a Super-complex, composed of CSN and Cullin-based E3 ligases (Rongere, thesis, 2004; Rongere, unpublished results, 2005) We hypothesized that Lats2 may perform its physiological function through interaction with CSN and Cullin-based E3 ligases. The present work on Lats2 has confirmed that Lats2 is a CSN associated kinase. We defined the domain specific interactions of Lats2 with hSocs3, hWsb1 (Sots box proteins) and hFBX-7 (F box protein), as well as the physiological consequences of interaction with hSocs3, hWsb1 and hSocs1. Both the N-terminal and the kinase domains of Lats2 interact with full-length hSocs3, hWsb1 and hFBX-7, determined in GST pull-down assays suggesting that full-length Lats2 protein is involved in interactions. Refinement of the Lats2 interaction with hSocs3 indicated that the kinase domain of Lats2 interacts with a region of hSocs3 containing a SH2 domain located upstream of the Socs box domain of the hSocs3. Moreover, Lats2 phosphorylated specific regions between the N-terminal and SH2 domain (S l) as well as between the SH2 domain and Socs box domain of hSocs3 (S3).These results indicate that hSocs3 is a novel Lats2 substrate. The kinase assay has also demonstrated that wt Lats2 was able to phosphorylate hSocs3, but not Lats2 kinase dead mutant (deleted ATP pocket, Lats20ATP). Mutational analysis identified two serine residues located at positions 144/145 (S3) to be specifically phosphorylated by wt Lats2. Phosphorylation of proteins has been shown to be a signal for proteolytic degradation of many characterized proteins. Thus we hypothesized that Lats2 could target hSocs3 for proteasomal degradation. When wt Lats2 was over-expressed in HEK293T cells and COST cells, the half-life of hSocs3, as a component of Elongin BC Cullin-based E3 ubiquitin ligase (EBC ligase), decreased significantly. In contrast, aver-expression of the Lats2OATP did not alter the half-life of hSocs3. Furthermore, the stability of hSocs3 depended on phosphorylation of serine residues at positions 144/145 by wt Lats2. Although the sites of phosphorylation were not defined for two other substrate recognition modules of EBC ligasehWsbl and hSocsl, their half-lives also decreased when wt Lats2 was over-expressed. To test in vivo, we synthesized esiRNA to knock-down endogenous Lats2 and subsequently we measured the half-lives of hSocs3 and hVVsb l . Here we demonstrated that the half-lives of hSocs3 and hWsbl were increased by the factor of two in Lats2-depleted HEK293T cells. In conclusion, our findings suggest that Lats2, a CSN associated kinase, is a novel regulator of EBC ligase function by regulating the turn-over of hSocs3, hSocs1 and hWsb1. Thus, Lats2 alters the specificity and capacity of EBC ligases regulating thereby the stability of numerous proteins which are targeted by EBC ligases for proteasomal degradation. Further studies should reveal whether the observed modulation of EBC ligase function by Lats2 associated with a Super-complex is also responsible for the turn-over of cell cycle regulators and the observed alteration in cell cycle by Lats2 over-expression.

HIV-1 co/super-infection in intravenous drug users.

BACKGROUND: The frequency of HIV-1 co/super-infection is unknown despite their implications for public health and vaccine development. This issue was addressed during an epidemic of both CRF11 and B subtype among intravenous drug users (IVDUs). METHODS: Bulk sequencing of reverse transcriptase, protease and C2V3 regions and subtype-specific nested polymerase chain reaction (PCR) in plasma and proviral DNA were performed using baseline and follow-up samples collected in recently infected IVDUs between 1998-2002 and in IVDUs with chronic infection living in the same area and presenting an unexpected rise of viremia (> 1 log10). RESULTS: In 58 recently infected patients, three B/CRF-11 co-infections, 25 B, 28 CRF-11 and two other subtypes were detected at baseline. In the three co-infected patients, both CRF-11 and B were detected in plasma and proviral DNA and persisted during follow-up. B- and CFR-11-specific PCR performed on follow-up samples of 40 of 58 recently infected patients (median follow-up, 14.5 months) revealed a transient B super-infection in a patient initially infected by CRF-11. Five of 156 chronic IVDUs (total follow-up: 346 years) had an unexpected rise of viremia. In two of them, aviremic without treatment for years after an initial B infection, a symptomatic CRF-11 super-infection occurred and was associated with high viral load and a fall of CD4 cell count. CONCLUSIONS: In recently infected IVDUs, co-infection B/CRF-11 is relatively frequent (5%). In chronically infected IVDUs super-infection may be transient and may occur in patients controlling efficiently HIV infection by the initial strain.

Large TCR diversity of virus-specific CD8 T cells provides the mechanistic basis for massive TCR renewal after antigen exposure.

Ex vivo analysis of virus-specific CD8 T cell populations by anchored PCR has shown that the CD8 TCR repertoire was less oligoclonal (seven to nine clonotypes per individual epitope) than previously thought. In the current study, TCR diversity was investigated by assessing both the overall TCR β-chain variable regions usage as well as the CDR3 regions in ex vivo-isolated CMV- and EBV-specific CD8 T cells from 27 healthy donors. The average number of clonotypes specific to most single viral epitopes comprised between 14 and 77. Changes in the CD8 TCR repertoire were also longitudinally assessed under conditions of HIV-1 chronic infection (i.e., in patients with suppressed virus replication and after treatment interruption and Ag re-exposure). The results showed that a large renewal (≤80%) of the TRB repertoire occurred after Ag re-exposure and was eventually associated with an increased T cell recognition functional avidity. These results demonstrate that the global CD8 TCR repertoire is much more diverse (≤9-fold) than previously estimated and provide the mechanistic basis for supporting massive repertoire renewal during chronic virus infection and Ag re-exposure.