Bioinformatics is a valuable tool to understand gene regulatory networks. Cis-regulatory elements (CREs) previously found in promoter regions are known to recruit transcription in signaling pathways. In this work it has been hypothesized to consider CREs as a family of related words that interact/bind to a family of related transcription factors, and thus have similar but distinct regulation patterns. A 1460 microarray gene expression collection was obtained via online databases to create a transcriptomic meta-dataset. A novel bioinformatic algorithm was applied to annotate all 65536 (64k) potential 8-letter CREs in the 500 bp upstream promoter region of all A. thaliana genes across the transcriptomic meta-dataset. Of the possible words, only 2,498 were significantly associated with a pattern of regulation in any of the 1,460 microarrays tested whereas the remaining motifs appeared not to be regulatory. Unique CREs were categorized into 4 regulatory types: inducer, suppressor, biregulator and insulator. A predicted protein protein interactome was created for an economically important plant Coffea canephora. Here, it has been hypothesized that evolutionary conservation of many core biological processes enable generation of predicted protein interactome for species with few resources other than sequenced genome. Of over 12,000 genes identified, 939 were predicted to have 4,587 interactions. Gene Ontology analysis revealed enrichment of processes conserved in all eukaryotes but depletion in unique plant processes. A third study was conducted to determine if homology modeling, evolutionary analysis, and structural evolution could determine key factors involved in function, and interaction specificity in Pus10 (EC 5.4.99.25) found in Archaea and Eukaryotes. Redundancy of Pus10 and the bacterial TrmA and TruB orthologs appear to have resulted in significant molecular evolution of Pus10 function. Neofunctionalization was identified in animal kingdom where thiouridine synthase, methylases and PSUSs (THUMP)-domain modification in early animal evolution coincides with appearance of TNF-related apoptosis-inducing ligand (TRAIL) apoptosis components. Subfunctionalization was identified for Thermococcales lineage of Archaea where a shorter forefinger-loop coincides with the loss of Ψ54 specificity as experimentally verified in P. furiosus. Absence of Pus10 was observed in Sulfolobus and higher fungi whereas in plant kingdom Pus10 function remains unknown with possible pseudogene in some lineages

The evolution of synapses from early proto-synaptic protein complexes in unicellular eukaryotes to sophisticated machines comprising thousands of proteins parallels the emergence of finely tuned synaptic plasticity, a molecular correlate for memory and learning. Phenotypic change in organisms is ultimately the result of evolution of their genotype at the molecular level. Selection pressure is a measure of how changes in genome sequence that arise though naturally occurring processes in populations are fixed or eliminated in subsequent generations. Inferring phylogenetic information about proteins such as the variation of selection pressure across coding sequences can provide valuable information not only about the origin of proteins, but also the contribution of specific sites within proteins to their current roles within an organism. Recent evolutionary studies of synaptic proteins have generated attractive hypotheses about the emergence of finely-tuned regulatory mechanisms in the post-synaptic proteome related to learning, however, these analyses are relatively superficial. In this thesis, I establish a scalable molecular phylogenetic modelling framework based on three new inference methodologies to investigate temporal and spatial aspects of selection pressure changes for the whole human proteome using protein orthologs from up to 68 taxa. Temporal modelling of evolutionary selection pressure reveals informative features and patterns for the entire human proteome and identifies groups of proteins that share distinct diversification timelines. Multi-ontology enrichment analysis of these gene cohorts was used to aid biological interpretation, but these approaches are statistically under powered and do not capture a clear picture of the emergence of synaptic plasticity. Subsequent pathway-centric analysis of key synaptic pathways extends the interpretation of temporal data and allows for revision of previous hypotheses about the evolution of complex synaptic function. I proceed to integrate inferred selection pressure timeline information in the context of static protein-protein interaction data. A network analysis of the full human proteome reveals systematic patterns linking the temporal profile of proteins’ evolution and their topological role in the interaction graph. These graphs were used to test a mechanistic hypothesis that proposed a propagating diversification signal between interactors using the temporal modelling data and network analysis tools. Finally, I analyse the data of amino-acid level spatial modelling of selection pressure events in Arc, one of the master regulators of synaptic plasticity, and its interactors for which detailed experimental data is available. I use the Arc interactome as an example to discuss episodic and localised diversifying selection pressure events in tightly coupled complexes of protein and showcase potential for a similar systematic analysis of larger complexes of proteins using a pathway-centric approach. Through my work I revised our understanding of temporal evolutionary patterns that shaped contemporary synaptic function through profiling of emergence and refinement of proteins in multiple pathways of the nervous system. I also uncovered systematic effects linking dependencies between proteins with their active diversification, and hypothesised about their extension to domain level selection pressure events.

Plasmodium falciparum (Pf) est un parasite intracellulaire capable d’infecter l’Homme. Dans les 48h après l’invasion des érythrocytes, il passe par le stade d’une cellule géante multinucléée qui se divise en 16 à 32 parasites. Cette multiplication rapide nécessite des mécanismes spécifiques de régulation très subtilement orchestrés. Parmi les modifications post-traductionnelles décrites chez les cellules eucaryotes, la phosphorylation réversible des protéines par les kinases/phosphatases est une des voies majeures dans la transduction des signaux cellulaires. Chez Plasmodium, des études de génétique inverse ont démontré que PfPP1, phosphatase majeure du parasite, est essentielle pour sa survie. L’activité de PP1 est connue pour être contrôlée par divers régulateurs endogènes. Cependant, malgré leur importance dans le ciblage de l’holoenzyme à un compartiment subcellulaire spécifique et/ou dans la régulation de son activité, peu de recherches ont été consacrées à l’identification de partenaires de PP1 chez Pf.Dans le but d’approfondir nos connaissances sur la régulation de PfPP1 et son impact sur la biologie de Pf, nous étudions les partenaires de cette enzyme qui seraient impliqués dans le contrôle de la phosphatase. Nos recherches récentes, basées sur des études de génomique comparative, ont permis d’identifier 4 régulateurs de PfPP1 : PfLRR1, PfI3, PfI2 et PfeiF2β. Au-delà de la capacité de ces protéines à contrôler la fonction de PP1 in vitro, nous avons montré par génétique inverse que leur rôle est vital pour Pf. En parallèle, nous avons entrepris une démarche plus globale pour la recherche de nouveaux partenaires/régulateurs de PfPP1. Nous avons notamment réalisé un criblage par double hybride de levure (Y2H) où PfPP1 est utilisé comme appât.Dans la 1ère partie de ma thèse, nous avons analysé les clones obtenus en criblage Y2H et initié la caractérisation de plusieurs d’entre eux. Notre choix d’étude s’est porté par la suite sur PF3D7_091900 et PF3D7_1202600, retrouvées 8 et 10 fois lors du criblage et présentant une interaction forte avec PP1. Mon projet de thèse avait pour but de caractériser au niveau moléculaire et fonctionnel le rôle de ces protéines sur l’activité de PP1 et de déterminer les régions/motifs de ces potentiels régulateurs pouvant intervenir au niveau de la relation structure/fonction du complexe qu’ils forment avec PfPP1.Dans une 2ème partie, nous avons étudié la séquence de ces protéines. PF3D7_0919900, protéine spécifique du parasite, possède le motif RVxF, souvent impliqué dans l’interaction avec PP1 et présente des motifs RCC1 connus pour interagir avec des protéines. Ainsi elle sera nommée RCC-PIP pour Regulator of Chromosome Condensation - Phosphatase Interacting Protein. PF3D7_1202600 est, quant à elle, un orthologue de Caliban chez la drosophile, et sera nommée CLP pour Caliban-Like Protein. Elle présente 17 motifs RVxF dont 7 se situent dans le fragment obtenu suite au criblage. Différentes approches ont confirmé que ces 2 protéines sont des partenaires de PfPP1. Cependant, la réalisation d’un test pNPP in vitro a mis en évidence une fonction activatrice de RCC-PIP, alors que CLP ne présente pas d’effet.Dans une 3ème partie, l’objectif était d’étudier plus en détails RCC-PIP. Nous avons démontré que le motif RVxF est impliqué dans l’interaction avec PfPP1. Puis nous avons étudié les motifs RCC1 et leur interactome par la réalisation d’un criblage Y2H en utilisant ces motifs comme appât. Une kinase a été trouvée (PfCDPK7) suggérant que RCC-PIP aurait un rôle de plateforme capable d’interagir avec 2 enzymes antagonistes. L’étude du rôle de RCC-PIP chez le parasite est actuellement en cours. La réalisation d’un knock-in a démontré l’accessibilité du locus. Un knock-out a également été effectué, mais l’absence d’intégration du plasmide indique que RCC-PIP serait essentielle au parasite. Pour confirmer cette observation, un knock-out conditionnel chez P. berghei est en cours de réalisation. / Plasmodium falciparum is an intracellular parasite that evolves in several stages of development in the vertebrate host. Within 48 hours after the invasion of erythrocytes, it goes through the stage of a multinucleated giant cell which divides into as many parasites as nuclei (16-32 parasites). This growth/fast division requires specific regulatory mechanisms subtly orchestrated. Among the post-translational modifications described in eukaryotic cells, the reversible phosphorylation of proteins by kinases/phosphatases is one of the major pathways in the cellular signal transduction. In Plasmodium, PP1 is predicted to catalyze the majority of protein dephosphorylation events, and has been shown to be essential in its survival using reverse genetic approaches. The activity of PP1 is known to be tightly controlled by various endogenous regulators. However, despite their importance in targeting the holoenzyme to a specific subcellular compartment and/or regulating its activity, little has been devoted to identify PP1 partners in the parasite.In order to deepen our understanding of the regulation of PfPP1 and its impact on the biology of Plasmodium, we study the partners of this enzyme that may be involved in the control of its location, its specificity and activity. Our recent research, based on comparative genomic studies, have identified 4 regulators of PfPP1: PfLRR1, PfI3, PfI2 and PfeiF2β. In parallel, since Plasmodium has a particular cell cycle and the function of PP1 should be adapted, we have undertaken a more global approach to the search for new partners/regulators of PfPP1 using different approaches including a yeast two-hybrid screening where PfPP1 was used as bait.In the first part of my thesis, the clones obtained in Y2H screening were analyzed and 2 clones were selected for further characterization. These clones, corresponding to PF3D7_091900 and PF3D7_1202600 were identified 8 and 10 times during the screening, a good indication about their expression in blood stages and their interactions with PfPP1 can be still detectable under high stringency conditions. Hence, my thesis project aimed to characterize molecularly and functionally of the role of these proteins on PfPP1. _x000D_In the second part, we have analysed the sequence of these two proteins. PF3D7_0919900, a parasite-specific protein, shows the canonical binding motif « RVxF », known to be involved in the interaction with PP1, and present on the fragment obtained following the screening. The sequence also has RCC1 motifs known to interact with proteins. Thereafter, this protein was designated as RCC-PIP for Regulator of Chromosome Condensation - Phosphatase Interacting Protein. As far as PF3D7_1202600 is concerned, it seems to be an ortholog of Caliban expressed by Drosophila, and designated Pf Caliban CLP-Like Protein. It has 17 potential RVXF binding motif, of which 7 are located in the fragment obtained following the screening. Different approaches such as GST pull-down or ELISA, identified these two proteins as partners of PfPP1. Using pNPP in vitro assay, we showed a slight activation of PfPP1 by RCC-PIP, while CLP had no effect.In the third part, the objective was to study in more detail the RCC-PIP. We showed that the RVxF motif is involved in the interaction with PfPP1. We then tied to identify the partners of RCC1 by screening a cDNA library of P. falciparum using RCC1 containing protein as bait. We showed that RCC-PIP is able to interact with a kinase (PfCDPK7) suggesting that RCC-PIP may act as a platform since it is able to interact with 2 enzymes with opposed activities. Analysis of the role of RCC-PIP in the parasite is currently underway. The production of a knock-in demonstrated the accessibility of the locus. A knock-out was also carried out, but the lack of integration of the plasmid suggests that RCC-PIP is essential to the parasite. To confirm this observation, a conditional knock-out in P. berghei is in progress.

Nociception is the sensory nervous system that detects harmful stimuli including excessive heat, cold, toxic chemicals, and noxious mechanical stimulations. Transient receptor potential (TRP) channels are a group of evolutionarily conserved ion channels consisting of 4 subunits, each with 6 transmembrane spans, and detect a variety of external and internal nociceptive stimuli. Due to their critical roles in nociception, it is essential to understand the mechanisms that regulate TRP channels and subsequent nociception. Here, I investigated two distinct types of regulation of Drosophila transient receptor potential cation channel A1 (TrpA1): regulation via the expression of different TrpA1 isoforms, and via its binding with associated proteins. I found that one of the TrpA1 isoforms, TrpA1(E), inhibits the thermal responses of other TrpA1 isoforms in vitro. I also identified potential TrpA1 binding partners through Co- immunoprecipitation (Co-IP) and mass spectrometry analysis. These binding partners need further validation and characterization through biochemical, cellular, and behavioral assays to illustrate their roles in nociception, and may serve as potential drug targets for chronic pain.

Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR

No description available.

Plasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics that target the parasite, but show low toxicity in the human host. Protein kinases, because of their integral roles in cell signaling networks, are considered to be attractive drug targets. Cyclin dependent kinases, or CDKs, and Mitogen-Activated Protein kinases, or MAPKs, are common to eukaryotes and regulate cellular processes of growth and proliferation. Plasmodium falciparum Protein Kinase 6, or PfPK6, is an atypical protein kinase that shows similarities to both MAPKs and CDKs. PfPK6 is expected to have an important role in the intraerythrocytic cell cycle progression and growth in the malaria organism, as it has been found to be essential in the parasite. In order to better understand the function of PfPK6 within Plasmodium, we have identified serveral potential substrates and interactors of the kinase using co-immunoprecipitation with an HA epitope-tagged cell line of PfPK6, as well as phosphoproteomic analysis. These methods resulted identification of 15 novel protein interactors, with 4 being studied for further investigation, and 45 putative substrates after strict peptide filtering, five of which are used in this study. In order to verify putative substrates and interactors, both in vitro and in vivo methods were used. In vitro kinase assays using GST-PfPK6 with 5 recombinant substrates confirmed direct phosphorylation of two novel substrates: MAL7P1.38, a regulator of chromosome condensation, and PF10_0047, a putative RNA binding protein. After attempts to generate bacterial constructs of several putative interactors and a global failure of a usable amount of protein to express under IPTG induction conditions, an alternative form of expression using a cell free Transcription and Translation reaction (TNT) with Wheat Germ Extract was used to generate radiolabeled PF11_0154, PFF0625w, and PF11_0305. Pull down analysis using GST-PfPK6 showed the kinase's ability to "pull" the interactors out of solution, confirming the interactions defined by the initial epitope tagged Co-Immunoprecipitation. Additionally, for in vivo analysis, parasites were transfected with RFP- PFF_0695w, an uncharacterized Plasmodium protein, in order to cellular localization of this interactors. Immunofluorescence assays of transfected lines showed punctate forms of PFF_0695w in the host erythrocyte in the late trophozoite and schizont stages of the parasite development, suggesting this interactor is a previously undiscovered protein in the Plasmodium secretome. The research presented here is an initial step to defining the interactome of PfPK6.

[ES] La activación de las diferentes cascadas de señalización en respuesta al estrés ambiental, así como mantener activas las proteínas y complejos proteicos en respuesta al estrés celular es fundamental para las plantas. La chaperona Hsp90 juega un papel importante en la coordinación de estos dos procesos, aunque los mecanismos que regulan su actividad en respuesta al ambiente no están completamente descritos. Estudios recientes en animales muestran que las proteínas prefoldin-like (PFDLs), co-chaperonas de Hsp90, desempeñan un papel importante en la señalización ambiental. Por lo tanto, son capaces de transmitir información sobre el medio ambiente para modular tanto el ensamblaje de complejos proteicos como parte del Hsp90-R2TP/PFDL como las vías de señalización en las que se encuentran involucradas. Hoy en día, se conoce muy poco sobre las proteínas PFDLs en especies vegetales. En este trabajo, hemos obtenido evidencia de que las PFDLs, particularmente URI1, pueden ejercer un papel similar en Arabidopsis, coordinando la homeostasis de las proteínas con las vías de crecimiento en respuesta a diferentes tipos de estrés, como por ejemplo el estrés por falta de energía. Así, mostramos que el complejo R2TP/PFDL se forma en Arabidopsis y que URI1 es una de sus subunidades. La actividad de URI1 es esencial para ciertos procesos, como el desarrollo embrionario, evidenciado por el arresto embrionario temprano causado por la mutación knock-out de URI1. Se ha observado que URI1 tiene una influencia notoria en el transcriptoma mediante el uso de un alelo hipomórfico de uri1. Coherentemente con lo observado en el transcriptoma, el interactoma de URI1 muestra que URI1 interactúa con un número relativamente grande de proteínas, muchas de las cuales están involucradas en procesos fundamentales relacionados con el metabolismo del ARN mensajero y la transducción de señales. URI1 es una proteína altamente versátil, aunque la base molecular de esta versatilidad aún es desconocida. Aquí mostramos que URI1 en Arabidopsis posee una región intrínsicamente desordenada que abarca la mayoría de la parte C-terminal de la proteína, característica que se conserva en los ortólodos de levadura y humanos. Nuestros resultados revelan en URI1 dos características principales de las proteínas desordenadas. La primera de ellas es la promiscuidad en las interacciones con otras proteínas y la segunda la inestabilidad de la proteína. Hipotetizamos que estas dos contribuyen a dotar a URI1 de versatilidad funcional. Es importante destacar que la inestabilidad de URI1 se contrarresta con el azúcar. El análisis genético realizado sitúa a URI1 en la vía de señalización que controla el crecimiento en respuesta al estrés energético inducido por la privación de azúcar, al desempeñar un papel como un regulador negativo aguas arriba de una de las quinasas principales, TOR. Hipotetizamos que URI1 desempeña un papel en la prevención del crecimiento excesivo de las plántulas cuando las condiciones energéticas son favorables. / [CA] És de vital importància per a una planta activar les corresponents cascades de senyalització en resposta a l'estrés ambiental i mantenir actives les proteïnes i els complexos proteics malgrat l'estrés cel·lular. La xaperona Hsp90, entre d'altres, s'encarrega de coordinar aquests dos processos, encara que els mecanismes que regulen la seua activitat en resposta a l'entorn no s'han arribat a comprendre del tot. Estudis recents en animals mostren que les co-xaperones de Hsp90, prefoldin-like (PFDLs), tenen un rol destacat en la senyalització ambiental. Per tant, tenen el potencial de portar informació sobre l'entorn per modular tant l'assemblatge de complexos proteics, com a part de l'Hsp90-R2TP/PFDL, i les vies de senyalització en les quals estan involucrats. Actualment, hi ha poca informació sobre PFDLs en espècies vegetals. Ara tenim l'evidència que els PFDLs, particularment URI1, poden exercir un paper similar en Arabidopsis, coordinant l'homeòstasi proteica amb les vies de creixement en resposta a l'estrés, per exemple, estrés energètic. En aquest treball mostrem que el complex R2TP/PFDL es forma a Arabidopsis i que URI1 és una de les seues subunitats. L'activitat d'URI1 és essencial per a alguns processos, com el desenvolupament embrionari, com es demostra per l'arrest precoç causat per la mutació knock-out d'URI1. Amb un al·lel hipomòrfic d'uri1, es va mostrar que URI1 té una forta influència en el transcriptoma. En consonancia amb l'observat amb el transcriptoma, l'interactoma d'URI1 mostra que URI1 interactua amb un nombre relativament gran de proteïnes, moltes de les quals estan involucrades en processos fonamentals relacionats amb el metabolisme de l'ARN missatger. Així, URI1 en Arabidopsis, com els seus ortòlegs en rent i humans, sembla estar involucrat en diverses funcions cel·lulars, incloent-hi l'homeòstasi proteica, el metabolisme del ARNm i la transducció de senyals. URI1 és una proteïna altament versàtil, encara que la base molecular d'aquesta versatilitat encara és desconeguda. Ací mostrem que Arabidopsis URI1 posseeix una gran regió intrínsecament desordenada que abasta la major part de la porció C-terminal de la proteïna, una característica que es conserva en els ortòlegs de rent i humans. Els nostres resultats revelen dues característiques principals de les proteïnes desordenades en URI1. La primera la promiscuïtat en les interaccions amb altres proteïnes, i la segona la inestabilitat proteica. Aleshores, hipotetitzem que aquestes dues característiques contribueixen a dotar URI1 de versatilitat funcional. Curiosament, la inestabilitat d'URI1 es contraresta amb el sucre, i la nostra anàlisi genètica situa URI1 en la via de senyalització que controla el creixement en resposta a l'estrés energètic induït per la privació de sucre, actuant com a regulador negatiu aigües amunt de la quinasa TOR. Hipotetitzem que URI1 exerceix un rol en prevenir el creixement excessiu de les plàntules quan les condicions energètiques són favorables. / [EN] It is of fundamental importance for the plant to trigger the corresponding signaling cascades in response to environmental stress and to keep proteins and protein complexes active despite the cellular stress. The chaperone Hsp90 plays an important role in coordinating these two processes, although the mechanisms that regulate its activity in response to the environment are not fully understood. Recent studies in animals show that the Hsp90 co-chaperones prefoldin-like (PFDLs) play a role in environmental signaling. Therefore, they have the potential to carry information about the environment to modulate both the assembly of protein complexes as part of the Hsp90-R2TP/PFDL and the signaling pathways in which they are involved. Currently, there is little information on PFDLs in plant species. We have now accumulated evidence that PFDLs, particularly URI1, may exert a similar, general role in Arabidopsis, coordinating protein homeostasis with growth pathways in response to stress, e.g. low energy stress. Here we show that the R2TP/PFDL complex is formed in Arabidopsis and that URI1 is one of its subunits. The activity of URI1 is essential for certain processes, such as embryonic development, as evidenced by the early arrest caused by the knock-out mutation of URI1. With a hypomorphic uri1 allele, URI1 was shown to have a strong influence on the transcriptome. Consistent with this, the URI1 interactome shows that URI1 interacts with a relatively large number of partners, many of which are involved in fundamental processes related to mRNA metabolism. Thus, Arabidopsis URI1, like its orthologs in yeast and humans, appears to be involved in diverse cellular functions, including protein homeostasis, mRNA metabolism and signal transduction. URI is a highly versatile protein, although the molecular basis of this versatility is still unknown. Here we show that Arabidopsis URI1 possesses a large intrinsically disordered region spanning most of the C-terminal portion of the protein, a feature that is conserved in yeast and human orthologs. Our results reveal two main features of disordered proteins in URI1: promiscuity in interactions with partners and protein instability. We hypothesize that these two features contribute to endowing URI1 with functional versatility. Interestingly, the instability of URI1 is counteracted by sugar, and our genetic analysis places URI1 in the signaling pathway that controls growth in response to sugar deprivation-induced energy stress by acting as a negative upstream regulator of the master kinase TOR. We hypothesize that URI1 plays a role in preventing excessive seedling growth when energy conditions are favorable. / La realización de esta Tesis Doctoral ha sido posible gracias a una Ayuda para Contratos Predoctorales para la Formación de Doctores FPI (BES-2017-081041) y una ayuda europea EMBO Scientific Exchange (9583). Asímismo, el trabajo experimental ha sido financiado por el proyecto [ILOVEPFD] del Ministerio de Ciencia e Innovación AEI-MICINN (PID2019-109925GB-I00). / Gómez Mínguez, Y. (2024). Characterization of URI1 from Arabidopsis Thaliana and its role in stress responses [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203592

Le cancer de la prostate (CaP) est devenu un véritable problème de santé publique dans les pays industrialisés. L'hormonothérapie reste le traitement de première ligne le plus efficace dans les cancers avancés mais il n'empêche pas la progression vers un stade androgéno-indépendant (AI), pour lequel la chimiothérapie s'avère peu efficace. Une des stratégies pour améliorer les thérapies actuelles consiste à cibler des gènes de survie surexprimés dans les CaPs AI afin de restaurer la sensibilité aux traitements du CaPs. Hsp27, protéine surexprimée dans ces cancers, à un effet cytoprotecteur qui engendre une résistance aux traitements. Elle est maintenant reconnue comme une cible thérapeutique importante. Rocchi et al. ont développé un oligonucléotide antisense (ASO) de deuxième génération (OGX-427) qui cible l'ARNm d'Hsp27. OGX-427 est actuellement en essai clinique phase II chez des patients atteints de CaPs au Canada et aux Etats-Unis. Mon projet de thèse a porté sur l'étude des mécanismes d'action d'Hsp27 impliqués dans l'évolution AI du CaP. Cette étude a pour but d'améliorer la sureté pharmacologique d'OGX-427, mais aussi d'identifier de nouvelles cibles thérapeutiques visant spécifiquement les cellules tumorales. Mes travaux de thèse ont montré que lors d'un stress cellulaire induit par hormonothérapie et/ou chimiothérapie, Hsp27 interagit avec le facteur eucaryotique d'initiation de la traduction eIF4E et le protège de sa dégradation par la voie ubiquitine/protéasome. Ceci maintient la synthèse protéique et engendre une survie cellulaire impliquée en partie dans l'effet cytoprotecteur médié par Hsp27. / Prostate cancer (PC) has become a real public health issue in industrialized countries, mainly due to patients' relapse by castration-resistant (CR) disease after androgen ablation. One strategy to improve current therapies in advanced PC involves targeting genes that are activated by androgen withdrawal, either to delay or prevent the emergence of the CR phenotype. Hsp27 is over-expressed in this cancer and has been shown to play a cytoprotective role leading to treatments resistance. This protein is now considered as promising therapeutic target. Rocchi, P. et al. developed and patented a second generation antisens oligonucleotides (ASO) targeting Hsp27 that has been licensed (OGX-427) and phase II clinical trials are currently in process in PC in Canada and USA. My PhD project focused on the study of Hsp27 action mechanisms involved in CRPC progression. The present study aims to improve pharmacological safety of OGX-427 and to identify new therapeutic targets specific of CRPC cells. The results of my PhD have shown that during cell stress induced by hormone- and/or chemotherapy, Hsp27 interacts with eukaryotic translation initiation factor eIF4E and protects it from degradation by the ubiquitin/proteasome pathway. This maintains protein synthesis and leads to cell survival, partly involved in the cytoprotection mediated by Hsp27. Our work therefore concerned the characterization of the interaction site between Hsp27 and eIF4E in order to identify potential inhibitors of this interaction that could delay CRPC progression.

Le cancer de la prostate (CaP) représente la deuxième cause de mortalité par cancer chez l'homme. La suppression androgénique (castration-thérapie) demeure la seule thérapie efficace du CaP avancé du fait de son caractère castration-sensible. Cependant, elle n'empêche pas la progression castration-résistante (CR) de la maladie dans les 1 à 3 ans après le début du traitement hormonal. Récemment, l'implication d'Hsp27 (Heat shock protein 27) dans l'échappement thérapeutique des CaPs a été montrée et un oligonucléotide antisens inhibiteur d'Hsp27, OGX-427, est en cours d'évaluation clinique II/III pour le traitement des CaPs CR. Afin de comprendre le rôle d'Hsp27 dans le mécanisme de résistance à la castration, nous avons réalisé le criblage de l'ensemble des protéines partenaires d'Hsp27 par double hybride. Mes travaux de thèse ont permis d'identifier une nouvelle protéine cliente d'Hsp27, TCTP (translationally controlled tumor protein) dont l'expression est indétectable dans les cellules normales. J'ai également montré que la progression CR des CaPs corrélait avec une surexpression de TCTP, une perte de P53 et que l'inhibition de TCTP par un oligonucléotide antisens restaurait l'expression de P53. Cette étude suggère, pour la première fois, un lien direct entre P53 et la sensibilité à la castration des CaPs. De plus, l'étude de l'interactome d'Hsp27 a mis en évidence son implication dans de nouvelles fonctions telles que la réparation de l'ADN ou l'épissage alternatif des ARNm. L'ensemble de ces travaux ont permis de mieux comprendre les mécanismes d'action d'Hsp27 dans la progression CR des CaPs et de développer de nouvelles approches thérapeutiques. / Prostate cancer (PC) is the second most common cause of cancer-related mortality in men in the Western world. Androgen ablation (castration-therapy) is usually the initial therapy in patients with advanced or metastatic disease. Unfortunately, the disease gradually progresses to a metastatic castration-resistant (CR) state, which remains incurable. Recently, the involvement of Hsp27 (Heat Shock Protein 27) in CR progression has been identified and an oligonucleotide antisense (OGX-427), inhibitor of Hsp27 is currently in phase II/III clinical trials to treat CRPC. In order to understand Hsp27 mechanisms of action in CR progression, we started to screen for Hsp27 partner proteins by using two-hybrid system. My PhD work has reported that Translationally Controlled Tumor Protein (TCTP) was a new Hsp27 protein partner that mediated Hsp27 cytoprotection in CRPC and that TCTP expression was absent in normal prostate tissues. We have further found that CR progression correlated with TCTP overexpression, the loss of P53 and that TCTP silencing using an antisense was able to restore P53 expression and function. This study suggests for the first time that castration-sensitivity is directly linked to P53 expression. In addition, we revealed exciting new aspects of the Hsp27 involvement in essential metabolic and cellular processes such as DNA repair and mRNA splicing. In summary, my PhD results have provided an enriched understanding of Hsp27 mechanisms of cytoprotection contributing to CRPC progression and opened a new promising field of research for multi-target therapeutic approaches.