Upon responding to cellular stress, p53 protein becomes stabilised and acts as a transcription factor mainly resulting from phosphorylation and acetylation of the protein. Nitration of p53 protein is poorly characterised by comparison with phosphorylation and acetylation. The main aim of this work was to study the effects of nitration on p53 functional activities and on p53-MDM2 protein-protein interactions. Preliminary work was to characterise the nitration of p53 protein over-expressed in E. coli BL21(DE3) which was then purified by a series of column chromatography. GST-MDM2 protein along with control GST protein were also overexpressed in BL21 which were subsequently purified by a single step batch purification before subjected to nitration. Peroxynitrite, a nitrating agent used in this study, was generated in vitro. Preliminary nitration work was carried out using BSA as a model protein as it is easily nitrated owing to its high number of tyrosine residues (19 residues). The present results showed that p53 and GST-MDM2 proteins were hardly nitrated as no strong nitro-tyrosine signals were obtained. This might be due to these proteins, being overexpressed in E. coli, were not properly folded resulting in hidden/cryptic tyrosine residues of which making nitration difficult to achieve. Peroxynitrite was shown to have a degrading property, reducing protein levels of peroxynitrite-treated p53, GST-MDM2 and GST proteins. Immunoprecipitation studies of cancer cell lysates with different p53 status treated with peroxynitrite showed very weak signals of nitro-p53 protein in mutant p53 cells whereby no nitro-p53 protein signal in wild-type p53 MCF7 cells. In addition, NO donor GSNO-treated MCF7 cells showed weak nitro-p53 protein signals. / Ministry of Science, Technology and Innovation (MOSTI) of Malaysia

The p53 tumour suppressor protein lies at the hub of a very complex network of cellular pathways including apoptosis, cell cycle arrest, DNA repair and cellular senescence. However, the mechanism of why and how p53 switches between apoptosis and cell cycle arrest, thereby determining a cell’s fate, remains a mystery to us. To enable us to investigate this ability of p53 to switch between cell cycle arrest and apoptosis, we developed a model which demonstrates similar p53 expression patterns but different functional outcomes. Treating cells with Cisplatin (a common chemotherapeutic drug) and Nutlin-3 (an MDM-2 inhibitor) results in similar high levels of p53 accumulation but different cellular responses. Cisplatin-treated cells undergo apoptosis while Nutlin-treated cells enter cell cycle arrest. Using this model, we explored the localization of p53 and in particular a C-terminal Ser 392 moiety in an attempt to identify how p53 is able to preferentially activate cell cycle arrest or apoptotic pathway.

La protéine suppresseur de tumeur p53 est soumise à de complexes régulations transcriptionnelles et posttraductionnelles. La découverte d’isoformes de p53 a introduit un degré de complexité supplémentaire auxmécanismes de régulation des fonctions de p53. On dénombre à ce jour douze isoformes qui diffèrent de p53dans leurs domaines N- et C-terminal. Cependant, les modes d’expression et de fonction de ces isoformes restentà être clarifiés.Dans cette thèse, nous nous sommes intéressés aux deux isoformes Δ40p53 et Δ133p53, en analysant leurinteraction avec p53 et en mesurant leur expression dans les mélanomes, un type de cancer où p53 est trèsrarement mutée. Nous montrons que Δ40p53 peut moduler l’activité de p53 avec un effet bi-phasique, tantôtactivateur ou répresseur du niveau d’expression et des fonctions de p53. Δ133p53 est produite par un promoteurP2 localisé dans le gène TP53. Nous avons montré qu’en réponse à un stress génotoxique, l’expression de Δ133p53 est régulée par p53, qui se lie au promoteur P2. Ceci suggère une boucle d’auto-régulation par p53, quiest capable de contrôler l’expression d’une isoforme inhibant ses propres fonctions. Enfin, les isoformes Δ40p53 et Δ133p53 sont surexprimées dans les tumeurs métastatiques de mélanomes comparées aux tumeurs noninvasives,suggérant à ces isoformes un rôle dans l’inactivation de p53 dans les cancers.Ainsi, Δ40p53 et Δ133p53 interagissent avec p53 de façon complexe, avec des effets plus contrastés que lasimple inhibition de l’activité suppressive de p53. Les isoformes de p53 jouent ainsi un rôle majeur dans lesactivités basales de p53, ainsi que dans l’inactivation fonctionnelle de p53 dans les cancers. / The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.

The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.