Mutation of Vav1 adaptor region reveals a new oncogenic activation

International audienceVav family members function as remarkable scaffold proteins that exhibit both GDP/GTP exchange activity for Rho/Rac GTPases and numerous protein-protein interactions via three adaptor Src-homology domains. The exchange activity is under the unique regulation by phosphorylation of tyrosine residues hidden by intra-molecular interactions. Deletion of the autoinhibitory N-terminal region results in an oncogenic protein, onco-Vav, leading to a potent activation of Rac GTPases whereas the proto-oncogene barely leads to transformation. Substitution of conserved residues of the SH2-SH3 adaptor region in onco-Vav reverses oncogenicity. While a unique substitution D797N did not affect transformation induced by onco-Vav, we demonstrate that this single substitution leads to transformation in the Vav1 proto-oncogene highlighting the pivotal role of the adaptor region. Moreover, we identified the cell junction protein β-catenin as a new Vav1 interacting partner. We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of β-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts. In addition, a similar interaction is evidenced in epithelial lung cancer cells expressing ectopically Vav1. In these cells, Vav1 is also involved in the modulation of β-catenin phosphorylation. Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity. INTRODUCTION The Vav1 proto-oncogene has a restricted hematopoietic expression and exhibits both GTP/GDP exchange activities (GEF) for Rho family GTPases and adaptor functions within signalling complexes [1, 2]. Two other genes, Vav2 and Vav3 belong to the same family of signalling effectors and share high structural similarities and properties with Vav1. Unlike Vav1, Vav2 and Vav3 have an ubiquitous expression [3, 4]. Vav proteins display a number of characteristic structural domains with homology for: Calponin (CH), Dbl (DH), Pleckstrin (PH) and Src (SH2 and SH3) altogether with acidic residues-rich (AcR) and cysteine-rich (CR) motives. These domains mediate interactions with membrane receptors

Human Dna2 is a nuclear and mitochondrial DNA maintenance protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance

The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed

Identification de ligands de quadruplexes de guanines inhibiteurs de l'activité télomérase

Les télomères de mammifères sont composés de répétitions en tandem TTAGGG auxquelles sont associées de nombreuses protéines qui protègent les extrémités des chromosomes. Le télomère, en plus de la région double-brin, se termine par une extension simple-brin 3'. Les télomères protègent les chromosomes et empêchent leur dégradation ou leur raccourcissement jusqu'à une longueur critique qui conduirait à l'altération de leur intégrité et à l'entrée de la cellule en sénescence. La télomérase est une enzyme spécialisée de type transcriptase inverse qui assure la réplication complète des chromosomes. La télomérase est très fortement exprimée dans les cellules cancéreuse et à un niveau très faible dans les cellules somatiques. Elle constitue donc une cible thérapeutique majeure en oncologie. L'extrémité simple-brin 3' peut adopter une structure particulière de l'ADN : le quadruplexe de guanines. La stabilisation d'un quadruplexe de guanines par des ligands spécifiques constitue une approche thérapeutique potentielle pour inhiber l'activité de la télomérase. Nous avons entrepris un criblage de banques de molécules pour identifier des ligands spécifiques qui stabilisent le quadruplexe de guanines en utilisant des méthodes biophysiques et biochimiques (FRET, dialyse à l'équilibre, essai TRAP). Plusieurs familles de ligands ont été identifiées : parmi elles, deux composés naturels (ascididémine et méridine) ; les dibenzophénanthrolines, les triazines et les pyridines dicarboxamides. En évaluant la sélectivité de ces molécules, nous avons montré que les pyridines dicarboxamides (avec la télomestatine) sont les ligands les plus sélectifs décrits à ce jour. Nous avons également montré que le traitement de cellules cancéreuses par certains dérivés triazines conduit à un arrêt de la prolifération cellulaire associé à un raccourcissement des télomères mais également à une entrée en sénescence.Mammalian telomeres are composed of TTAGGG repeat arrays and associated proteins that function to protect and maintain telomeric DNA. In addition to the double-strand region, telomeric DNA consists of a 3' overhang. In normal human cells telomeric DNA progressively erodes with each round of cell division due to insufficient levels of telomerase, a specialized reverse transcriptase necessary to fully replicate the chromosomal ends. Telomerase in highly expressed in tumor cells but at very low levels in most somatic cells and thus telomerase is an attractive target for the development of novel chemotherapeutics. The 3' overhang is prone to adopt a G-quadruplex structure and the stabilisation of this structure bi ligands inhibits telomerase activity. We focused on the screening and the identification of specific dyes that stabilize G-quadruplex structure and inhibit telomerase activity using biophysical and biochemical methods (FRET, equilibrium dialysis, TRAP assay). Several families have been identified like natural compounds (meridine and ascididemine), dibenzophenanthrolines, triazines and pyridine dicarboxamides. We evaluated the selectivity of these dyes and we have shown that pyridine dicarboxamides derivatives (with telomestatin) are the more selective G-quadruplexes ligands described so far. We also have shown that G-quadruplexes ligands such as triazine derivatives lead to a cell proliferation arrest that is associated with telomere shortening and senescence in cancer cells.PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Télomères et Télomérase : des cibles toujours pertinentes en oncologie ?

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G-Quadruplex Recognition by Quinacridines: a SAR, NMR, and Biological Study

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Régulation de la longueur des télomères : rendre le télomère accessible ?

International audienceLes extrémités des chromosomes linéaires, ou télomères, sont à l'état normal protégées de tout événement indésirable de fusion. L'altération de leur structure participe à l'instabilité génétique, tandis que l'érosion des répétitions télomériques à chaque division constitue un mécanisme de contrôle de la proliféra-tion à long terme des cellules somatiques. Alors que la structure de la télomérase humaine est assez bien connue, les partenaires qui régissent la stabilité et la conformation de son substrat, l'extrémité télomérique, le sont moins. En particulier, ce n'est que très récemment que l'on a établi le lien fonctionnel entre les protéines liant la partie double brin du télomère et celles fixant l'extrémité simple brin 3' sortante riche en guanine. Cette revue a donc pour objectif de faire le point sur les facteurs qui contrôlent l'allongement des télomères et les modèles actuels de régulation. ▲ Abstract. Under a normal state, the extremities of chromosomes, telomeres, are protected against undesired fusion events. Alterations of the telomere structure are associated with genetic instability, while erosion of the telomeric repeats, occurring at each cell division, provides a mechanism controlling the long-term proliferation of somatic cells. Although the structure and composition of the human telomerase enzyme are now well characterized, the protein partners regulating the stability and conformation of its DNA substrate, the telomeric end, are much less known. A functionnal link has been recently evidenced between proteins that bind the double-stranded telomere repeats and those recruited at the 3' G-rich telomeric overhang. This review presents an update on these telomeric factors controlling telomere lengthening and discuss the actual models proposed for its regulation.