The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket.

No description available.

HIPK1 (Homeodomain Interacting Protein Kinase 1) est une sérine/thréonine kinase de la superfamille des CMGC kinases. Les HIPKs, initialement identifiées comme des régulateurs des facteurs de transcription à homéodomaine, constituent un groupe de quatre kinases. Parmi elles, trois semblent jouer un rôle dans la modulation de l’activité de p53. Il est bien connu que HIPK2 peut phosphoryler p53 sur sa sérine 46 en activant sa fonction pro-apoptotique et plus récemment il a été découvert que HIPK4 pouvait phosphoryler p53 sur sa sérine 9. Concernant HIPK1 le rôle fonctionnel de son interaction avec p53 n’a pas encore été clairement élucidé. De plus, la régulation d’autres voies de signalisation, telles que celle des MAP kinases par HIPK1 a été aussi documentée. L’objectif de mes travaux a été de caractériser la fonction de HIPK1 dans les cellules cancéreuses afin d’identifier son rôle potentiel dans la biologie tumorale, i.e. dans les processus de cancérogénèse ou dans la réponse aux traitements. Par sur-expression de HIPK1 nous avons caractérisé sa localisation subcellulaire, identifié ses déterminants moléculaires, et trouvé que HIPK1 est rapidement dégradée dans le compartiment nucléaire. De plus nous avons trouvé que HIPK1 active principalement la transcription p53-dépendante, en phosphorylant p53 sur sa sérine 15 et en stabilisant cette dernière. La sur-expression de HIPK1 a un effet anti-prolifératif, associé avec l’induction de p21. Nous avons donc exploré le profil d’expression de HIPK1 dans une collection de 80 cancers colorectaux et observé que HIPK1 est souvent plus fortement exprimée dans les tissus tumoraux que dans les tissus sains, mais aussi que son expression est indépendante du statut de p53 (sauvage ou muté). / HIPK1 (Homeodomain Interacting Protein Kinase 1) is a serine/threonine kinase which belongs to the CMGC kinases superfamily. HIPKs that have been initially identified as regulators of homeodomain transcription factors constitute a group of four kinases. Among them, three seem to play a role in the modulation of p53 activity. While it is well known that HIPK2 can phosphoylate p53 on serine 46 and activates its proapoptotic function, it has more recently been shown that HIPK4 can also phosphorylate p53 on serine 9. Concerning HIPK1, although some first studies have indicated that it could interact with p53, the functional role of this interaction has not been clearly elucidated. Moreover, the regulation of other signaling pathways, such as MAP Kinases by HIPK1 has also been documented. The purpose of this work is to characterize HIPK1 function in cancer cells to identify its possible role in tumor biology, i.e. in cancerogenesis process or in the response to treatments. By overexpressing HIPK1 we characterized the subcellular localization of the kinase, identified its molecular determinants, and found that HIPK1 is rapidly degradated in the nuclear compartment. Moreover we found that HIPK1 activates principally the p53 dependent transcription, by phophorylating p53 on its serine 15 and by stabilizating it. The overexpression of HIPK1 has an anti-proliferative effect, associated with the induction of p21.We have explored the HIPK1 expression profile in a collection of 80 colorectal cancers and observed that HIPK1 is often strongly expressed in the tumor tissue compared to the normal tissue, but also that its expression is p53 status independent (wild-type or mutated).

La sénescence réplicative (SR) dans les fibroblastes humains primaires est causée par l’érosion des télomères et est contrôlée par p53. La régulation dynamique de l’activation de p53 est essentielle pour l’induction de la sénescence ; cependant, les mécanismes moléculaires sous-jacents ne sont pas clairement établis. Nous montrons, dans les cellules surexprimant les isoformes Δ133/Δ160p53, que ces isoformes s’oligomérisent avec p53, conduisant ainsi à la stabilisation d’une forme inactive de p53. A l’inverse, l’inactivation des isoformes endogènes Δ133/Δ160p53 induit l’accumulation de la protéine p53 et l’activation de son activité transcriptionnelle. La surexpression de Δ133/Δ160p53 inhibe les fonctions de p53, en particulier son activité transcriptionnelle et son rôle dans l’arrêt du cycle après un dommage à l’ADN. Nous avons remarqué que les protéines Δ133/Δ160p53 et p53 sauvage possédaient des conformations différentes. Les protéines Δ133/Δ160p53 sont reconnues pas l’anticorps Pab40 : elles adopteraient une conformation similaire à un mutant de conformation de p53. Enfin, nous observons qu’une faible expression de l’ARNm Δ133/Δ160TP53 coïnciderait avec la durée de l’activation transcriptionnelle de p53 lors de la SR, indiquée par l’accumulation de l’ARNm d’un effecteur majeur de p53, p21. L’augmentation de l’expression de Δ133/Δ160TP53 à un temps tardif au cours de la SR est corrélée à l’accumulation du marqueur de sénescence p16INK4a et à celle de la cytokine pro-inflammatoire IL-6. En conséquence, les isoformes Δ133/Δ160p53 contrôleraient l’activité de p53 dans l’arrêt du cycle et sur le phénotype sécrétoire des cellules sénescentes. / Telomere attrition in primary human fibroblasts induces replicative senescence by activation of the tumour suppressor p53. Fine-tuned activation of p53 is essential for senescence induction; however, the mechanisms underlying the regulation of p53 activity during senescence have not been clearly established yet. We report here that in cells that express the Δ133/Δ160p53 isoforms, these p53 isoforms oligomerize with p53, leading to the stabilization of the transcriptionally inactive form of p53. Conversely, endogenous Δ133/Δ160p53 silencing increases the level of p53 and p53-dependent transcriptional activity to promote cell cycle arrest. Overexpressed Δ133/Δ160p53 repress p53 functions, including gene transcription activation and growth inhibition, upon DNA damage. We also found that Δ133/Δ160p53 and wild-type p53 have different structural conformations. Δ133/Δ160p53 adopt a more unfolded conformation recognized by the Pab240 antibody, indicating that these p53 isoforms have a p53 mutant-like conformation. Finally, we observed that low level of Δ133/Δ160TP53 mRNA coincided with the duration of p53 transcriptional activation in replicatively senescent fibroblasts, as indicated by the upregulation of CDKN1A (p21) mRNA expression, a downstream effector of p53. Δ133/Δ160p53TP53 was upregulated at a later stage when the senescence marker p16INK4a and the pro-inflammatory interleukin-6 (IL-6) were also induced. Therefore, p53 activity on growth suppression and senescence-associated secretory phenotype may be differentially regulated by its Δ133/Δ160p53 isoforms.

Damages to the genetic materials arise throughout the lifespan of a cell, and elicit upregulation of DNA repair factors. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is part of a DNA repair protein complex that specialises in the repair of DNA base modifications and single-strand breaks (SSBs). TDP1 removes a broad spectrum of chemical adducts from the 3' end of a DNA strand break, including topoisomerase 1 (TOP1) peptide, during DNA transcription and replication. Inactivation or deletion of TDP1 is associated with cerebellar dysfunction and degeneration, with remarkably little extraneurological manifestation. The reason for the selective dependence of the cerebellar neurons on TDP1 activity is not clear. It was hypothesised that the TDP1 activity is upregulated in tissues with high levels of SSBs, either from DNA transcriptional activity, or reactive oxygen species (ROS)-induced damage. The aim of this doctoral project was therefore to identify and characterise the cellular mechanisms that regulate TDP1 activity. Our lab has previously shown that the Nterminus domain (NTD) of TDP1 covalently interacts with DNA ligase 3α. In this thesis, evidence has been presented to show that this interaction is regulated by the putative ATM/ATR/DNA-PK phosphorylation site, serine 81, to prolong TDP1 half-life, and enhance cellular survival after genotoxic stress. A second post-translational modification in the NTD by SUMOylation of the K111 residue was identified, enlightening a mechanism by which TDP1 is recruited to sites of transcription-mediated SSBs. To investigate the requirement for TDP1 in cells under high levels of oxidative stress, I have developed a mouse cellular model whereby the levels of endogenous ROS can be modulated by overexpression of the human anti-oxidant enzyme superoxide dismutase 1 (SOD1) or its toxic mutant SOD1G93A. Overexpression of SOD1G93A in Tdp1-/- MEFs induces accumulation of chromosomal SSBs and decreases survival after H2O2 challenge, while overexpression of SOD1 has a protective effect. Besides repair of ROS-induced TOP1-cc in the nucleus, TDP1 also repairs mitochondrial topoisomerase 1-mediated DNA breaks. This role is required during transcription and assembly of mitochondrial subunits of the electron transfer chain complexes, and has direct impact on mitochondrial respiration and ROS production. Collectively, these data provide mechanistic insights into regulation of TDP1-mediated chromosomal and mitochondrial DNA repair.

The tumour suppressor p53 is a key regulatory protein that prevents proliferation of damaged cells. Under unperturbed conditions, the ubiquitin ligase murine double minute 2 (MDM2) mediates p53 ubiquitination and further degradation by the proteasome. In consequence p53 is present at low levels, but becomes rapidly stabilised and activated in response to a variety of stimuli, such as DNA damage or virus infection. P53 responds to these diverse stresses to regulate the expression of many target genes that induce cell cycle arrest, DNA repair, or apoptosis. The attenuation of p53 interaction with MDM2 is maintained by enzymes catalysing p53 post-translational modifications such as phosphorylation. Casein kinase 1 α (CK1α) is one such enzyme; it stimulates p53 after DNA virus infection. Surprisingly depletion of CK1α using small interfering RNA or inhibition using a CK1 kinase inhibitor activated the transcription factor p53, indicating that p53 steady-state level is controlled by CK1α. Disrupting MDM2-p53 interaction using small molecule Nutlin-3 displayed similar pharmacological properties to the CK1 inhibitor on p53, indicating that the MDM2-CK1α complex co-regulates p53 stability. Indeed co-immunoprecipitation of endogenous CK1α with MDM2 occurred in undamaged cells. CK1α was shown in vitro to directly bind to and phosphorylate MDM2. Therefore it appears that CK1α must be recruited into specific complexes under different conditions, which can influence its substrate selectivity and explain its dual role on the p53 pathway. Apart from CK1, there are few other kinases whose action can directly contribute to the inhibition of p53. A novel pyrazolo-pyridine analogue showing dual activity against CK1 and Checkpoint kinase 1 led to increased p53 activation. These data highlighted the potential value of dual kinase inhibitors as therapeutics in cancer. The dominant protein-protein interface that stabilises the MDM2-CK1α complex was mapped using a peptide-based approach. One CK1α peptide bound strongly to MDM2, it specifically disrupted the protein-protein interaction, and its transfection was able to reduce cancer cell growth. A peptide phage display approach was finally combined with Next-Generation Sequencing to define the change in MDM2 binding motifs when the CK1α peptide or Nutlin-3 is bound, compared to ligand-free MDM2, and thus will help to understand protein-protein interaction network re-wirings which led to cell growth inhibition.

1 / Lucia Jie Chiao

Erlangen, Nürnberg, Universiẗat, Diss., 2007. / Enth. 1 Sonderabdr. aus: International journal of radiation biology ; Vol. 83. 2007. - Beitr. teilw. dt., teilw. engl.

Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. Thioredoxin reductase specifically catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin provides a source of electrons for disulfide reduction in various cellular processes. Reduction of disulfides within the cell can be accomplished by the separate but partially overlapping glutathione reductase - glutathione - glutaredoxin pathway. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Δtrr1 yeast lacking the gene encoding thioredoxin reductase. The Δtrr1 mutation caused an increased in oxidation in both molecules. Highcopy expression of the GLR1 gene encoding glutathione reductase in Δtrr1 yeast restored the redox state of glutathione to wild-type levels, but did not restore p53 activity. Also, p53 activity was unaffected by be a Δglr1 mutation, even though the mutation was known to result in glutathione oxidation. These results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment. In order to test if p53 activity requires an intact thioredoxin system in mammalian cells, dominant-negative and RNAi approaches designed to suppress thioredoxin reductase activity were used in a breast adenocarcinoma cell which contains an endogenous wild-type p53. In cells stably transformed with a plasmid encoding a dominant-negative form of thioredoxin reductase, thioredoxin reductase activity was inhibited 4.3-fold and p53 reporter gene expression was inhibited by 2-fold. In cells stably transformed with a RNAi plasmid designed to target thioredoxin reductase mRNA, thioredoxin reductase activity was inhibited by 1.7-fold and p53 reporter gene expression was inhibited by 1.6-fold. A decrease in the protein levels of the p53 endogenous target genes p21 and Bax was also observed in both dominant-negative and RNAi transformants. Additionally, thioredoxin was shown to bind p53 in vitro (Kd=0.9 μM), and a LexA-thioredoxin fusion protein was shown to bind p53 in vivo. These results suggest that p53 activity is regulated by thioredoxin reductase in mammalian cells through a direct interaction with thioredoxin. / Graduation date: 2004

ROGDI was a novel gene with unknown function, located on human chromosome 16p13.3. The coding region of the gene is 864 bp that encodes 287 amino acids. According to GenBank database, ROGDI contains leucine zipper domain. Previous studies in our laboratory showed that ROGDI increases cell proliferation in cell lines. In addition, overexpression of ROGDI induces p53 and p27 mRNA levels in human glioma cell line T98G and U251. In this study, we use two hepatocellular carcinoma cell lines, Hep G2 and Hep 3B, which contains wild-type and deleted tumor suppressor protein p53 respectively to investigate the expression of p53 and the response of anticancer drugs treatment in ROGDI overexpression cells. In addition, we compare the relation between the cell apoptosis the expression of p53 and ROGDI. Hence, we found that expression of p53 and ROGDI influences the cell response to anticancer drugs and induces apoptosis.