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.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24971号 / 医科博第153号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

P53 est un facteur de transcription qui se décline, chez l’homme, la souris ou la drosophile, en plusieurs isoformes. Chez la drosophile deux isoformes ont été caractérisées : la forme canonique Dp53 ; et une forme tronquée, D∆Np53, dont le domaine de transactivation est incomplet. Une des questions encore peu étudiée, concerne les mécanismes par lesquels p53 régule une grande variété de réponses cellulaires lors d’un stress. Pour répondre à cette question, nous avons étudié le rôle des isoformes de p53 dans la régulation de deux mécanismes antagoniste en lien avec la maladie de Parkinson (MP) : l’autophagie et l’apoptose en réponse à un stress délétère et un stress hormétique du système nerveux. Nous avons montré que les drosophiles portant une mutation nulle de p53 sont plus sensibles aux effets du paraquat (fort stress oxydant, modèle chimique de la maladie de Parkinson). En absence de p53, ce stress cause une forte inhibition de l’initiation et du flux de l’autophagie accompagné d’une augmentation des niveaux de caspases et de la mortalité. L’augmentation de la mortalité et des niveaux de caspases est similaire chez des mutants de l’autophagie pour lesquels le flux d’autophagie est constitutivement altéré. D’autre part, nous avons montré que les deux isoformes de p53 (Dp53 et D∆Np53) régulent différemment l’apoptose et l’autophagie dans les neurones photorécepteurs de drosophile : l’isoforme Dp53 présente un flux autophagique fonctionnel retardant la neurodégénérescence, tandis que, l’isoforme D∆Np53 inhibe le flux d’autophagie via l’activation des caspases Dcp1, Drice et Dronc. Enfin, nous avons établi un lien entre p53 et le stress du réticulum endoplasmique (RE). Dans un premier temps nous avons montré qu’un stress modéré du RE (pré-conditionnement) a un effet protecteur dépendant de l’activation de l’autophagie dans différents modèles de la MD. Ensuite, nous avons montré que les trois banches de la réponse au stress du RE (IRE1, Atf6 et PERK) sont impliquées dans cet effet protecteur. Enfin, nous avons montré que les drosophiles mutantes pour p53 perdent l’effet protecteur du pré-conditionnement. L’ensemble de nos résultats apporte de nouveaux éléments sur l’aspect multifonctionnel de p53 en réponse à un stress dans le système nerveux. Via ses multiples isoformes, p53 peut activer deux réponses antagonistes : l’autophagie et l’apoptose, permettant aux cellules une réponse flexible face à une situation de stress. La régulation de l’autophagie par p53 est protectrice et apparait comme étant une fonction ancestrale de p53. / P53 is a tumor suppressor gene, which has been showed to regulate several cellular pathways. Upon stress, p53 triggers multiple cellular pathways including DNA repair system, cell cycle arrest, apoptosis and autophagy. Thus, p53 is involved in both cellular protection and death pathways. One of the major questions is to understand how a single protein can promote so many different pathways. Here I address the putative role of p53 isoforms in the regulation of autophagy and apoptosis and their role in neuron survival in the context of Parkinson’s disease (PD). We show that p53 mutants are more susceptible to paraquat toxicity (chemical model of PD), indicating a protective role for p53. We also found that Atg8 mutant, which display an impaired autophagy, behave similarly to p53 upon paraquat treatment. In addition, we show that p53 is required for the activation of autophagy with a functional autophagy flux upon paraquat treatment and that lack of p53 or Atg8 results in an accumulation of activated caspases after paraquat treatment. Moreover, we found that autophagy and apoptosis were differentially regulated by different p53 isoforms. The Dp53 (p53B) isoform induced protective autophagy, whereas the D∆Np53 (p53A) isoform inhibited autophagy by activating the caspases Dronc, Drice and Dcp-1 in differentiated neurons. Our results demonstrate that a combination of the differential use of p53 isoforms and the antagonism between apoptosis and autophagy favors the generation of an appropriate p53 biological response to stress. In addition, we have defined in vitro and in vivo experimental conditions in which the activation of the Unfolded Protein Response (UPR) does not induce cell or organism lethality but rather promotes an adaptive response that protects from apoptotic stimuli. We show that this mild activation, known as ER-preconditioning is protective in several models of PD in an autophagy-dependent fashion. We showed that the three branches of the UPR are involved in the protective effect induced by ER-preconditioning. We then demonstrated that p53 is necessary to mediate the protection by ER-preconditioning suggesting that p53 may be a key factor in the integration of stress responses. Together our results reveal new aspects of the multi-functionality of p53. Activation of the antagonist pathways: autophagy and apoptosis by p53 isoforms, leads to flexible and adaptive response to stress. In addition, our results suggest that the regulation of autophagy by p53 is a ancestral protective function of p53.

p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.