Rozprawy doktorskie na temat „SUMO Protease”

Sumoylation has emerged as an important player in several biological processes involved in the maintenance of genome stability. A number of proteins implicated in DNA metabolism-associated processes, such as DNA replication, repair and chromosome segregation, were shown to sumoylated. Moreover, alterations in protein sumoylation and desumoylation, caused by deregulation of enzymes involved in the SUMO pathway, were associated with cancer development. Work done previously in our laboratory and by other groups suggested an important role for sumoylation in promoting DNA damage tolerance. However, the molecular mechanisms by which DNA damage regulates sumoylation and how balance between sumoylation and desumoylation impinges the cellular response to genotoxic stress are questions that need to be clarified. Different scenarios could account for regulating sumoylation, one of which envisages a control over the activity of the desumoylating enzymes. Budding yeast has two desumoylating enzymes, Ulp1 and Ulp2, which have distinct cellular localization and enzymatic activities. Starting by investigating whether Ulp1 and/or Ulp2 undergo any type of functional regulation, I found the nuclear SUMO protease Ulp2 to be phosphorylated both in a cell cycle-dependent manner and in response to replication stress or DNA damage. The present work aimed at studying the molecular mechanisms through which Ulp2 promotes genome integrity, both in physiological conditions and in conditions of replication stress. By examining the genome-wide localization of Ulp2 by ChIP-on-chip I observed that Ulp2 is bound to centromeric regions throughout the cell cycle and this enrichment is extended to pericentromeric regions in cells arrested at metaphase. Lack of Ulp2 affects the chromatin structure at centromeres and nearby regions. Following replication stress, Ulp2 is bound to most of the origins of replication that are active under our experimental conditions and is required for efficient origin firing. Also under conditions of replication stress Ulp2 is important for the maintenance of chromatin structure and for the binding of histone variants that are known to play a role in damage tolerance to origins of replication. Thus, Ulp2 appears to play key roles in regulating the status of chromatin to make it suitable for efficient replication and chromosome segregation.

PML et les corps nucléaires (CN) sont impliqués dans la défense antivirale. En effet, notre équipe a montré que la surexpression de PMLIII confère la résistance au virus de la stomatite vésiculaire, au virus de l'influenza, au virus foamy mais pas au virus de l’encéphalomyocardite (EMCV). J’ai montré dans mon travail de thèse que l’EMCV contrecarre le pouvoir antiviral de PMLIII en induisant sa dégradation par un processus dépendant du protéasome et de SUMO. Cependant, les cellules de souris invalidées pour PML sont plus sensibles à l’infection par l’EMCV que les cellules issues de souris parentales. Pour déterminer l’isoforme de PML responsable de cet effet antiviral, j’ai analysé l’effet des sept isoformes de PML (PMLI-VII) et j’ai montré que seule l’expression en stable de PMLIV confère la résistance à l’EMCV en séquestrant la polymérase virale 3Dpol au sein des CN PML. De plus la déplétion de PMLIV augmente la production de l’EMCV dans les cellules traitées par l’interféron. Ces données indiquent le mécanisme par lequel PML confère la résistance à l’EMCV et révèlent que PML est l’une des protéines médiatrices des effets anti-EMCV de l’interféron<br>PML and nuclear bodies (NBs) are implicated in antiviral defense. Indeed, our team showed that overexpression of PMLIII confers resistance to vesicular stomatitis virus, influenza virus, foamy virus but not to encephalomyocarditis virus (EMCV). I have shown during my thesis that EMCV counteracts the antiviral effect of PMLIII by inducing its degradation in SUMO and proteasome-dependent way. However, cells derived from PML knockout mice are more susceptible to EMCV infection than wild-type cells. To determine the isoforme of PML implicated in this antiviral effect, I analysed the effect of the seven PML isoforms (PMLI-PMLVII) and I showed that only stable expression of PMLIV confers resistance to EMCV by sequestring the viral polymérase 3Dpol in PML Nbs. In addition, depletion of PMLIV boosted EMCV production in interferon-treated cells. These finding sindicate the mechanism by which PML confers resistance to EMCV and reveal a new pathway mediating the antiviral activity of interferon against EMCV

Au sein des cellules, les ARNms sont liés par de nombreuses protéines, générant ainsi des particules appelées mRNPs (Ribonucléoparticules de messagers). Leur formation est cotranscriptionnelle, et leur composition va réguler l’ensemble des étapes du métabolisme des ARNms : stabilité, maturation, export, localisation et traduction. Au vu de l’importance de ces mécanismes dans la physiologie cellulaire, le contenu protéique des mRNPs est finement régulé dans le temps et l’espace et fait l’objet de nombreux remodelages. Ces changements de composition dépendent notamment des hélicases, ainsi que des modifications post-traductionnelles ; cependant, ces mécanismes demeurent à caractériser de façon plus approfondie. Une modification post-traductionnelle susceptible de moduler ces remaniements depuis la levure S. cerevisiae jusqu’aux métazoaires est la sumoylation. En effet, la SUMO-protéase Ulp1/SENP2, une enzyme clé de la machinerie de sumoylation, est localisée au panier des pores nucléaires, à proximité d’une plateforme d’ancrage des mRNPs destinées à l’export. Par ailleurs, il a été rapporté chez la levure que des mutants affectant la localisation et la stabilité d’Ulp1 présentent des défauts d’export et de localisation des mRNPs. Au vu de ces données, le laboratoire s’est intéressé aux rôles potentiels de la sumoylation dans le métabolisme de ces particules d’ARNm. Dans ce but, un crible protéomique a été réalisé chez la levure S. cerevisiae afin de comparer la composition des mRNPs entre des cellules sauvages ou mutantes pour Ulp1. Ce crible a mis en évidence un rôle d’Ulp1 dans le recrutement de deux composants des mRNPs, le complexe THO et l’hnRNP Hek2. Le complexe THO est un facteur multiprotéique qui participe à la prévention de l’instabilité génique et contribue à la transcription des ARNms, à l’assemblage des mRNPs et à leur export. L’hnRNP Hek2 est une protéine aux rôles multiples, dont l’association à un ARNm est susceptible de moduler sa stabilité, sa traduction et/ou sa localisation. Des analyses biochimiques nous ont permis de mettre en évidence l’existence de formes sumoylées de la sous-unité Hpr1 du complexe THO ainsi que de l’hnRNP Hek2. Toutes deux sont Ulp1-dépendantes, et interviennent sur la partie C-terminale de ces protéines. Nous avons également mis en évidence que chacune de ces sumoylations contrôle le recrutement de son substrat au sein des mRNPs. L’analyse fonctionnelle d’un mutant affectant la sumoylation d’Hpr1 a identifié cette modification comme nécessaire au recrutement du complexe THO sur une population d’ARNms impliqués dans la résistance au stress acide, autrement dégradés par l’exosome. Ainsi, l’absence de sumoylation d’Hpr1 diminue fortement la viabilité cellulaire en conditions de stress, un phénotype supprimé par l’inactivation de l’exosome. L’étude des effets de la sumoylation d’Hek2 suggère une modulation par SUMO de certaines de ses fonctions, notamment dans la localisation cellulaire des ARNms. L’ensemble de ces données fournit donc les deux premiers exemples de régulation du métabolisme des mRNPs par des événements de sumoylation intervenant au niveau du pore nucléaire<br>Within the cells, mRNAs are associated to proteins, thereby generating particles called mRNPs (messenger ribonucleoproteins). mRNPs form in a cotranscriptional manner and their composition defines the fate of mRNAs by modulating the different steps of their metabolism, including their stability, their processing, their export, their localisation and their translation. In view of the importance of such mechanisms for cell physiology, several mechanisms ensure a tight spatio-temporal control of mRNPs composition through multiple mRNP remodelling events. These changes in the protein content of mRNPs depend on helicases and post-translational modifications, but remain to be further investigated. Sumoylation is one of the modifications that could contribute to mRNPs remodelling from yeast (S. cerevisiae) to metazoans. Indeed, it has been reported that the SUMO-protease Ulp1/SENP2, a key enzyme of the sumoylation machinery, is localized at the basket of nuclear pore complexes, in close vicinity with mRNPs committed for export. This particular localization, together with the reported defects in mRNPs export and localisation of yeast mutants affecting Ulp1, prompted the lab to ask whether sumoylation could contribute to mRNP biogenesis. In order to investigate this hypothesis, our lab compared mRNPs composition between wild-type and ulp1 mutant S. cerevisiae yeast strains using a proteomic approach. This screen identified two mRNP components that depend on Ulp1 for their recruitment onto these particles: the THO complex and the hnRNP Hek2. The THO complex is a multi-subunit factor that prevents genome instability and contributes to transcription, mRNP assembly and export. Hek2 has multiple functions in mRNA stability, translation and/or localization. Using biochemical approaches, we have been able to visualize sumoylated versions of the Hpr1 subunit of the THO complex and of the hnRNP Hek2. In both cases, this modification depends on Ulp1 activity and occurs on the C-terminal part of the protein. We further showed that these sumoylation events control THO and Hek2 recruitment onto mRNPs. Functional analysis of a mutant impairing Hpr1 sumoylation revealed that this modification is required for proper recruitment of the THO complex onto a subset of mRNAs involved in acidic stress resistance, which are otherwise degraded by the exosome. Decreased Hpr1 sumoylation results in a strong reduction of viability in acid stress conditions, a phenotype that is rescued by inactivation of the exosome. The investigation of the role of Hek2 sumoylation in mRNPs metabolism suggests that this modification regulates some of Hek2 functions, especially in mRNA localisation. All together, these results provide the two first examples of mRNPs components whose functions are regulated by sumoylation events occurring at the level of nuclear pores

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1′) of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1′ glutamate (Glu379) with an arginine residue (Arg302) of the β-sheet C. A P1′ glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1′ residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin–protease interaction. Arg302 is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1′ Glu379, which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu379 and Arg302 influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

Intercambiar la especifidad de las isoformas de SUMO1 y SUMO2/3 para SENP6/SENP7 SENP6 y SENP7 son los miembros más divergentes de la familia de proteasas SENP y los únicos miembros que llevan cuatro inserciónes o “loops” localizadas en su dominio catalítico. Al sobreponer el dominio catalítico de SENP7 sobre el complejo SENP2-SUMO podemos ver una posible interfaz entre el Loop1 de SENP7 y SUMO. También identificamos diferentes residuos de SUMO1 y SUMO2 que podrían formar parte de la interfaz. Diseñamos una serie de mutantes de SUMO1 y SUMO2 donde se intercambian entre ellos los residuos que forman parte de la interfaz. De esta manera fuimos capaces de intercambiar la actividad proteolítica de SENP6 y SENP7 hacia estos substratos. El Loop1 de SENP6 y SENP7 es responsable de la especificidad de la interfaz entre los SUMOs y las SENP6/7 Diseñamos una serie de mutantes del Loop1 de SENP7 dentro de la posible interfaz entre el Loop1 de SENP7 y SUMO para determinar los papeles estructurales y funcionales de los residuos que están dentro de esta región. Los mutantes de SENP6/7 sin el Loop1 perdían gran parte de su actividad, esta se podía recuperar un poco al reemplazar las cuatro prolinas del Loop1 por glicinas. De esta manera probamos que el Loop1 tiene un papel por lo menos estructural en el reconocimiento de SUMO. También identificamos el residuo Lys691 del Loop1 de SENP7 como elemento indispensable en la actividad de la enzima ya que al mutar este residuo por un aspártico disminuye la actividad para sustratos con SUMO2. Por otra parte el residuo Asp71 de SUMO2 es uno de los residuos propuestos para el reconocimiento de SUMO2/3. Cuando mutamos este residuo a lisina, vemos una bajada en la actividad de SENP7. Este hecho sugiere que Asp71 de SUMO2 está interaccionando con Loop1 y quizas con la Lys691, y que la bajada en la actividad es causada por una repulsión de cargas. Por otro lado con el objectivo de estudiar el efecto de Loop1 en la desconjugación de especies SUMOyladas, insertamos los ocho residuos de SENP6 Loop en el dominio catalítico de SENP2. Esta inserción provoca un aumento de la actividad sustancial de SENP2 contra diSUMO en comparación con la forma wild type. Complejos con substratos Para ver si SENP6 es capaz de formar algún complejo estable, producimos cantidades en miligramos de los mutantes inactivos de Δ3SENP6C1030S y de Δ2Δ3SENP6C1030S. Incubamos cada proteasa con los precursors de SUMO, con RanGAP1-SUMO2 y con diSUMO2. De todos los substratos que probamos, diSUMO2 fue el único que podía formar un complejo estable con Δ3SENP6CS y Δ2Δ3SENP6CS por copurificación en una columna de gel fitración. Con Δ2SENP6CS no pudimos formar ningún complejo. Con esta información llegamos a la conclusión que el Loop3 de SENP6 reduce, quizá por razones de entropía, la capacidad de SENP6 de formar un complejo estable con diSUMO2. Caracterización del Loop3 de SENP6 Para descifrar el papel que este loop tiene en el contexto de la proteasa, producimos y purificamos los 185 residuos de Loop3 de SENP6. Análysis 1-H 1-D RMN mostraba una falta de estructura terciaria dentro del loop, pero proteólisis limitada y espectroscopia de masas mostraban un fragmento estable de cerca de 11kDa. Dicroísmo circular y FTIR tambien sugieren la presencia de algunos elementos de estructura secundaria. Globalmente, la caracterización biofísica del Loop3 de SENP6 muestra que el loop es una proteína desestructurada.<br>Swapping the SUMO Isoform Specificity of SENP6/7 SENP6 and SENP7 are the most divergent members in the SENP family of proteases and they are the only members that bear four loop insertions dispersed throughout their catalytic domains. The superposition of the SENP7 catalytic domain with the SENP2-SUMO complex revealed a tentative SENP7 Loop1-SUMO interface and upon further inspection, distinct residues on SUMO1 and SUMO2 were identified at the interface. A series of mutants were constructed bearing characteristics of both SUMOs and by swapping the residues from SUMO1 to SUMO2 and vice versus we were able to both decrease and increase the activity of the SENP6 and SENP7 toward these substrates. Loop1 SENP6/7 Is Responsible For SUMO Interface Specificity In addition to mutations on SUMO we constructed a series of mutations on SENP7-Loop1 within the tentative SENP7-Loop1-SUMO interface to determine the structural and functional roles of the residues that reside within this region. We were able to recover some of the activity lost by the removal of Loop1 by replacing the four prolines of Loop1 with glycines proving that Loop1 plays at least a structural role in SUMO recognition. We also identified Lys691 of Loop1 in SENP7 as indispensible to the activity of the enzyme. D71 is one of the residues proposed to confer SUMO2/3 specificity in the previous swapping experiments. We mutated this residue in diSUMO2 and saw a decrease in activity of SENP7. This could be explained by our theory that this region on SUMO is interacting with Loop1, more specifically K691, and the decrease in activity was caused by a charge clash between SUMO2 and SENP7 Loop1. To further show the utility of Loop1 in deconjugation of multi-SUMOylated species we inserted the eight residues of SENP6 Loop1 into SENP2. We saw an overall increase in activity of SENP2 against diSUMO2 but not against any other substrate tested. Complexes With Substrates In order to see if SENP6 was able to form any stable complexes in solution, we produced milligram amounts of Δ3SENP6CS and Δ2Δ3SENP6CS (the two constructs of the protein that showed both good yields in protein production and high performance in activity assay). We incubated each protease with SUMO precursors, RanGAP1-SUMO2 and diSUMO2 substrates. Of all the substrates tested, only diSUMO was able to form a stable complex with Δ3SENP6CS and Δ2Δ3SENP6CS. Δ2SENP6CS was also tested but there was no indication of any complex formation, leading to the hypothesis that SENP6 Loop3 was impeding, perhaps entropically, the ability of SENP6 to form a stable complex with diSUMO2. SENP6 Loop3 Characterization Loop3 takes up roughly 40% and 20% of the catalytic domains of SENP6 and SENP7 respectively. In our loop deletion experiments we saw an overall increase in the activity when Loop3 was not present and removal of Loop3 proved vital to the ability of the enzyme to form a stable complex with diSUMO2. In order to try to decipher what role this loop plays in the context of the protease, we isolated the 184 insert from SENP6 and produced and purified the protein. 1-H 1-D NMR pointed to an overall lack of tertiary structure within the loop but limited proteolysis and mass spectrometry analyses showed a stable fragment of around 11kDa. Further circular dichroism and Fourier Transform Infrared Spectroscopy suggested the presence of some secondary structural elements but overall characterization of SENP6 Loop3 showed a mainly unstructured loop.

Plants, as primary produces of energy and the main source of food for many organisms, are both ecologically and economically important to sustaining life. Understanding the molecular mechanisms by which plants respond to stress is key to optimizing food production and maintaining biodiversity in an increasingly uniform global ecosystem. Arabidopsis thaliana is the major model plant species in which primary research provides insights into the workings of more important plant species. The research presented here advances our understanding of the role of SUMOylation and deSUMOylation in plants. The discovery of a previously undescribed group of SUMO proteases in plants, called deSUMOylating Isopeptidases (DeSis) has provided evidence for the first time of SUMOylation cycles outside the nucleus. Using proteomic comparisons from animal species, this group of SUMO proteases has been classified into 3 distinct sub-classes; DeSi1, DeSi2 and the plant specific DeSi3. Characterisation of the SUMO isopeptidase activity was achieved through biochemical assays in which SUMO1 conjugation chains were reduced in the presence of the DeSi proteins. Furthermore, investigation into the role of the DeSi3 protein At1g47740 (DeSi3a), has revealed key regulation of the immune response pathway. DeSi3a knockout plants were more resistant to pathogen attack, showed a higher response to the detection of pathogenic elicitor molecules and appeared to be primed for immune response. Together this provided evidence that DeSi3a is a major negative regulator of plant immunity. In addition to this, investigation into potential DeSi3a target substrates has revealed that the activation of the key receptor for the perception of bacterial pathogens, FLS2, is SUMOylation dependent. Further evidence suggests that the fungal pathogen receptor, CERK1, may also be regulated by SUMO. This thesis provides evidence that the regulation of pathogen perception is SUMOylation dependent and that DeSi3a is a key negative regulator of defence.

Les facteurs de transcription NFAT (facteur nucléaire des cellules T activées) jouent un rôle physiologique important dans le développement et le fonctionnement de nombreux organes, notamment dans le système immunitaire et le système nerveux. Par conséquent, leur dérégulation a été impliquée dans diverses maladies humaines telles que le cancer, les maladies neurodégénératives et les maladies auto-immunes. La régulation de l'activité de NFAT par translocation nucléo-cytoplasmique a été largement étudiée. En revanche, la régulation du niveau protéique de NFAT par le système ubiquitine-protéasome est encore mal comprise. Pourtant, les protéines NFAT ont une durée de vie courte et la régulation de leur stabilité est donc essentielle pour le contrôle de leur activité.Dans une étude précédente, mon groupe a montré que l'E3 ubiquitine-ligase Trim17 se lie à NFATc3 mais ne favorise pas son ubiquitination et tend plutôt à stabiliser la protéine. Les résultats préliminaires obtenus suggéraient que Trim39, un partenaire de Trim17, pourrait être une E3 ubiquitine-ligase pour NFATc3 et que la SUMOylation de NFATc3 modulait sa stabilité. L'objectif de ma thèse était donc de comprendre les mécanismes par lesquels Trim39, Trim17 et SUMO régulent la stabilité de NFATc3.Au cours de ma thèse, j'ai caractérisé Trim39 comme une E3 ubiquitine-ligase de NFATc3. En effet, mes résultats indiquent que la surexpression de Trim39, mais pas de son mutant inactif, induit l'ubiquitination de NFATc3 dans les cellules. En revanche, la déplétion de Trim39 endogène diminue le niveau d'ubiquitination de NFATc3. La protéine Trim39 recombinante induit directement l'ubiquitination de NFATc3 in vitro. De plus, la surexpression de Trim39 diminue les niveaux protéiques de NFATc3 alors que la déplétion de Trim39 les augmente. J'ai également montré que Trim17 inhibe l'ubiquitination de NFATc3 induite par Trim39, à la fois dans les cellules et in vitro. Trim17 agit à la fois en réduisant l'activité E3 ubiquitine-ligase intrinsèque de Trim39 et en empêchant l'interaction entre NFATc3 et Trim39. En outre, j'ai montré qu'un mutant de NFATc3 ne pouvant être SUMOylé est moins ubiquitiné et plus stable que la forme sauvage de NFATc3, ce qui suggère que la SUMOylation de NFATc3 est importante pour son ubiquitination et sa dégradation. En outre, j'ai identifié un motif d'interaction à SUMO (SIM) dans la séquence de Trim39, par lequel Trim39 lie les polymères de SUMO2. La mutation de ce SIM dans Trim39 ou des sites consensus de SUMOylation dans NFATc3 diminue l'interaction entre Trim39 et NFATc3, et l'ubiquitination de NFATc3 induite par Trim39. Ces résultats suggèrent fortement que Trim39 reconnaît et ubiquitine préférentiellement les formes SUMOylées de NFATc3 et agit donc comme une « E3 ubiquitine-ligase guidée par SUMO » (STUbL) pour NFATc3. Enfin, nous avons mesuré l'impact de ces mécanismes sur la fonction physiologique de NFATc3. J'ai tout d'abord montré que Trim39 diminue l'activité transcriptionnelle de NFATc3. En outre, à l'aide de cultures primaires de neurones granulaires du cervelet, nous avons montré que la mutation des sites de SUMOylation de NFATc3 et la déplétion de Trim39 endogène aggravent l'apoptose neuronale, probablement en stabilisant la protéine NFATc3. En conclusion, l’ensemble de mes données indiquent que Trim39 module l'apoptose neuronale en agissant comme une STUbL pour NFATc3 et en contrôlant sa stabilité<br>NFAT (Nuclear factor of activated T cells) transcription factors play important physiological roles in the development and function of many organs, notably in the immune system and nervous system. As a consequence, their dysregulation has been implicated in various human diseases such as cancer, neurodegenerative diseases, and auto-immune diseases. The regulation of NFAT activity by calcium-dependent nuclear-cytoplasmic shuttling has been extensively studied. In contrast, the regulation of NFAT protein level by the ubiquitin-proteasome system is still poorly understood. However, NFATs are short-lived proteins and regulation of their stability is critical for controlling their activity.In a previous study, my group has shown that the E3 ubiquitin-ligase Trim17 binds NFATc3 but does not promote its ubiquitination and rather stabilizes it. Preliminary results suggested that Trim39, a partner of Trim17, might be an E3 ubiquitin-ligase for NFATc3 and that SUMOylation of NFATc3 might modulate its stability. Therefore, the goal of my PhD was to understand the mechanisms through which Trim39, Trim17, and SUMO regulate the stability of NFATc3.During my PhD, I have characterized Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, my results indicate that overexpression of Trim39, but not its inactive mutant, induces the ubiquitination of NFATc3 in cells. In contrast, silencing of endogenous Trim39 decreases the ubiquitination level of NFATc3. Recombinant Trim39 directly induces the ubiquitination of NFATc3 in vitro. Moreover, overexpression of Trim39 decreases the protein levels of NFATc3 whereas the silencing of Trim39 increases it. I have also shown that Trim17, which can bind Trim39, inhibits Trim39-mediated ubiquitination of NFATc3, both in cells and in vitro. Trim17 acts by both reducing the intrinsic E3 ubiquitin-ligase activity of Trim39 and by preventing the interaction between NFATc3 and Trim39. Furthermore, I found that a SUMOylation-deficient mutant of NFATc3 is less ubiquitinated and more stable than the wild type NFATc3, suggesting that SUMOylation of NFATc3 is important for its ubiquitination and degradation. Importantly, I identified one SUMO interacting motif (SIM) in the sequence of Trim39 through which Trim39 binds SUMO2 polymers via one of these SIMs. Mutation of this SIM in Trim39 or SUMOylation consensus sites in NFATc3 decreased the interaction between Trim39 and NFATc3, and the ubiquitination of NFATc3 mediated by Trim39. These results strongly suggest that Trim39 binds and ubiquitinates preferentially the SUMOylated forms of NFATc3 and therefore acts as a SUMO-targeted E3 ubiquitin-ligase (STUbL) for NFATc3. Finally, we have measured the impact of these mechanisms on the physiological function of NFATc3. I first found that Trim39 decreases the transcriptional activity of NFATc3. Furthermore, using primary cultures of cerebellar granule neurons as a model, we have shown that the mutation of the SUMOylation sites of NFATc3 and silencing of endogenous Trim39 enhances neuronal apoptosis, probably by stabilizing the NFATc3 protein. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a STUbL for NFATc3 and by controlling its stability

Il carcinoma della vescica è uno dei tumori di più frequente riscontro da parte dell’urologo, rappresentando la seconda principale causa di decesso fra tutte le neoplasie del tratto genitourinario. Più del 90% delle neoplasie maligne della vescica è rappresentato dai carcinomi delle cellule uroteliali. La scarsità di procedure non invasive attendibili per la diagnosi precoce, cui va aggiunta la complessa eterogeneità biologica che questo tumore riscontra nella pratica clinica, sono alla base dei decennali tentativi della comunità scientifica nell’individuazione di biomarcatori tumorali, configurabili come indicatori precoci dell’esistenza del processo neoplastico, da utilizzare anche per la sua sorveglianza a lungo termine. Alla luce di tali considerazioni, la necessità di identificare nel tumore vescicale alcuni indicatori biochimici e genetici altamente specifici, sensibili e valutabili, oltre che su frammenti di tessuto, anche su liquidi biologici (urine e plasma), è tutt’oggi al centro di numerosi studi scientifici. Lo scopo di questo lavoro è stato quello di analizzare l’espressione della serina proteasi HtrA1, che è nota fungere da soppressore tumorale in diversi tumori solidi, nel tessuto vescicale uroteliale umano in condizioni fisiologiche e tumorali, al fine di valutarne una eventuale alterazione dei livelli in presenza di neoplasia. Inoltre, abbiamo voluto estendere lo studio all’analisi dei fluidi biologici e alla valutazione di un possibile coinvolgimento dell’HtrA1 nella progressione della malattia. Infatti, studi più o meno recenti hanno dimostrato come l’HtrA1 sia una molecola in grado di esercitare una azione di controllo sulla crescita e proliferazione cellulare e di indurre la morte cellulare stimolando l’apoptosi. Sono stati reclutati per lo studio pazienti affetti da neoplasia vescicale uroteliale a diverso grado e stadio, soggetti sani e con cistite. Di ciascun individuo, sono stati raccolti campioni bioptici tissutali assieme ad urine e plasma. Gli studi di immunoistochimica da noi condotti hanno dimostrato che l’HtrA1 è una molecola espressa dall’urotelio della vescica in condizioni fisiologiche ed in patologie infiammatorie come la cistite batterica. Al contrario, la proteina è risultata assente in tutti i casi esaminati di carcinoma uroteliale con diverso grado di malignità e a differenti stadi di infiltrazione, fin dalle fasi più precoci di comparsa visibile della neoplasia. Una diversa espressione dell’HtrA1 tra i tessuti patologici ed i tessuti sani, nonostante simili livelli del trascritto, è stata evidenziata dall’analisi western-blotting, dalla quale è emersa la presenza di due forme dell’HtrA1, una nativa di peso molecolare di ~50 kDa ed una, che si origina per autoproteolisi della prima, di ~38 kDa. Solo la forma a più basso peso molecolare ha mostrato una diminuzione significativa in tutti i tessuti patologici analizzati rispetto ai sani, dimostrandosi idonea ad essere considerata un buon biomarcatore tumorale. Poiché questa proteina fu originariamente descritta come una proteasi di secrezione, abbiamo ipotizzato che essa potesse essere secreta da parte dell’urotelio nella cavità vescicale o dal tessuto nel sangue. Abbiamo quindi esaminato la presenza dell’HtrA1 anche nelle urine e nel plasma di tutti i soggetti reclutati, dimostrando un incremento significativo della proteina nell’urina e nel plasma dei pazienti con carcinoma rispetto ai soggetti sani. Il presente lavoro ha quindi evidenziato l’HtrA1 quale possibile marker tissutale e urinario/plasmatico utile nella diagnosi del carcinoma uroteliale della vescica. Inoltre, dati di biologia molecolare supportati dai risultati ottenuti in vivo ci hanno suggerito che, anche nella vescica umana, l’HtrA1 può assumere il ruolo di soppressore tumorale e che probabilmente sia l’urotelio normale adiacente a quello tumorale a determinare un aumento dell’HtrA1 nelle urine dei soggetti con carcinoma piuttosto che l’urotelio interessato dalla neoplasia, forse come risposta di protezione alla progressione della malattia.<br>Bladder cancer is one of the cancers most commonly encountered by the urologist, making it the second leading cause of death among all cancers of the genito-urinary tract. More than 90% of malignant neoplasms of the bladder is represented by the carcinomas of the urothelial cells. The lack of reliable non-invasive procedures for early diagnosis, together with the complex biological heterogeneity that this tumor has in clinical practice, are the basis of decades of efforts of the scientific community in identifying cancer biomarkers, configurable as early indicators of the existence of the neoplastic process, to be used also for its long-term surveillance. In the light of these considerations, the need to identify some highly specific and sensitive biochemical and genetic markers in bladder cancer, to be valued, as well as in tissue fragments, even in biological fluids (urine and plasma), is still the focus of numerous scientific studies. The purpose of this work was to analyze the expression of serine protease HtrA1, which is known to act as a tumor suppressor in various solid tumors, in human urothelial bladder tissue under physiological and neoplastic conditions, in order to assess a possible alteration of its levels in presence of cancer. In addition, we wanted to extend the study to the analysis of biological fluids and evaluation of possible involvement of HtrA1 in the progression of the disease. In fact, more or less recent studies, showed how the HtrA1 is a molecule capable of exerting a control action on cell growth and proliferation and to induce cell death by stimulating apoptosis. We recruited for the study patients with urothelial bladder cancer at different grade and stage, healthy subjects and with cystitis. Of each individual, tissue biopsy samples were collected along with urine and plasma. The immunohistochemical studies carried out showed that HtrA1 is a molecule expressed in bladder urothelium under physiological conditions and in inflammatory diseases, such as bacterial cystitis. On the contrary, the protein was absent in urothelial carcinoma with different degree of malignancy and at different stages of infiltration, right from the earliest stages of visible appearance of the neoplasm. A different expression of HtrA1 between the pathological and normal tissues, despite similar levels of the transcript, was detected by Western blotting, which revealed the presence of two forms of HtrA1, a native form with the molecular weight of ~ 50 kDa and another, which originates by autoproteolysis from the native one, of ~ 38 kDa. Only the HtrA1 form with lower molecular weight showed a significant decrease in all analyzed pathological tissues compared to the healthy counterparts, proving to be suitable to be considered a good cancer biomarker. Since this protein was originally described as a secreted protease, we hypothesized that it might be secreted by the urothelium in the bladder cavity or by tissue into blood. Thus, we examined the presence of HtrA1 also in the urine and plasma of all patients enrolled, demonstrating a significant increase of the protein in the urine and plasma of cancer patients compared to healthy subjects. The present work has therefore shown that HtrA1 may be considered a possible tissue and urinary/plasma biomarker, useful in the diagnosis of urothelial carcinoma of the bladder. In addition, data of molecular biology supported by the results obtained in vivo have suggested that, even in the human bladder, the HtrA1 can assume the role of tumor suppressor and that, probably, the normal urothelium adjacent to the tumor is responsible of the increase of HtrA1 in the urine of patients with carcinoma rather than the urothelium affected by cancer, perhaps as a protective response to disease progression.

Enzymatic, covalent attachment of the Small Ubiquitin-like Modifier (SUMO) protein to a substrate protein, SUMOylation, is a stress inducible post-translational modification conserved throughout eukaryotes. SUMO conjugation to proteins alters protein interactions, regulating signalling pathways in the cell, and modulating response. SUMO proteases process SUMO into its mature form as a prerequisite to conjugation, in addition to providing reversibility to the SUMOylation pathway by cleaving SUMO from substrate proteins. Salicylic acid (SA) is a key hormone in propagating defense activation and signalling against biotrophic pathogens in plants. An investigation into the role of SUMO proteases OVERLY TOLERANT to SALT1 and -2 (OTS1 and -2) in SA regulation was performed using Arabidopsis thaliana mutants and transgenic over expressing lines. OTS1 and -2 were required for the restriction of SA biosynthesis and signalling in unchallenged plants. Further, SA treatment promoted OTS1 degradation and accumulation of SUMO conjugates, suggesting a positive relationship between SUMO conjugation and SA synthesis. Mutants of the SUMO E3 ligase SAP and MIZ1 (SIZ1) possess reduced levels of SUMO conjugates whilst displaying elevated SA content and activated defenses. This apparent contradiction was investigated using single siz1 and triple ots1 ots2 siz1 mutants, which were found to possess comparable SA related phenotypes to the ots1 ots2 double mutant. Finally it was concluded that there is more to the regulation between SA biosynthesis and SUMOylation than the presence or absence of SUMOylated proteins, and further, that promotion of SUMO conjugates by SA may facilitate modulation of other signalling pathways.

Thèse (Ph.D.) -- Université de Montréal, 2004.<br>"Thése [sic] présentée à la Faculté des études supérieures en vue de l'obtention du grade de Philosophiae Doctor (Ph.D.) en chimie" Version électronique également disponible sur Internet.

Covalent attachment of the fusion protein, Small Ubiquitin-like modifier (SUMO), to recalcitrant target proteins (especially transmembrane proteins) make purification of the target protein much easier by enhancing its recombinant overexpression, protecting from degradation and improving protein folding, solubility and stability. After purification of the SUMO-tagged protein, SUMO protease (Ulp1), which specifically cleaves the SUMO-tag, is required to regenerate the native target protein. However, the known SUMO protease enzyme from Saccharomyces cerevisiae i.e., S.ce. Ulp1 enzyme is not very efficient, and the cleavage reaction requires many hours of incubation with the SUMO-tagged protein at room temperature or above for partial cleavage of the SUMO-tag. Under such conditions, many target proteins, especially the integral membrane proteins, get denatured and inactivated. Herein, we aim to provide a solution to this problem by discovering and producing psychrophilic (cold-active) SUMO proteases, which will deSUMOylate the SUMO-tagged protein efficiently at low temperature thus keeping the target protein stable and active. To reach this goal, we have successfully overexpressed and purified two novel psychrophilic SUMO proteases- C.ps. Ulp1 enzyme and M.an. Ulp1 enzyme from psychrophilic yeasts, identified through bioinformatics studies. Comparative activity studies performed with different SUMO-tagged proteins have suggested that M.an. Ulp1 has better activity than the S.ce. Ulp1. Further, bioinformatics tools have been utilized to explore the probable reasons for better activity observed for M.an. Ulp1 enzyme in certain cases compared to S.ce. Ulp1 enzyme.

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepreneurship<br>Unpredictable environmental changes have a negative impact on plant development. To respond to these abiotic and biotic stresses, plants adapted by improving intricate molecular mechanisms, including post-translational modifications (PTMs) that are responsible for modifying proteins and consequently modulating their activity. An important PTM is the fast and reversible attachment of the SUMO peptide, known as sumoylation. It is well reported that sumoylation controls several essential processes in plant development and stress responses. The SUMO attachment is carried out in four steps involving several components such as ubiquitin-like proteases (ULPs), SUMO activating enzyme (E1), SUMO conjugating enzyme (E2) and SUMO ligases (E3). In this pathway, the E1-E2-E3 enzymatic cascade is responsible for the attachment of the SUMO to the target protein. The SUMO proteases ULPs are involved in the maturation of SUMO in an early step of the pathway, but are also responsible for desumoylation or SUMO removal from the target proteins, and therefore for balance of SUMO-conjugate levels. Plant genomes encode for more ULPs than remaining components of the conjugating pathway and, adding to the multitude of phenotypes revealed by ULP mutants, they are considered a source of specificity. However, several plant ULPs lack functional characterization, including ULP2a and ULP2b. Early evidences associated ULP2a and ULP2b with plant developmental regulation and numerous abiotic stress responses. Due to the importance of these proteins, several strategies and molecular tools were developed to study ULP2 function and find possible targets. In the present work SUMO-His fusion transgenic plants were introgressed with ulp2a/b mutant for future functional studies and target purification/determination. Constructs for BiFC assays were produced and tested to study sumoylation dynamics and SUMO-interactions in plants. Finally, plant Pds5 orthologous proteins, named Pds5-Like Members (PLMs) were indicated bioinformatically as potential ULP2s targets, and were used here as a case study. PLM homozygous mutant plants were genotyped, their expression level determined, and they were characterized morphologically and in response to osmotic stress. PLMs were cloned for future protein purification and in vitro desumoylation assays. The present work will aid in the study of ULP2 function and amplify our knowledge about sumoylation in plants.<br>Alterações ambientais imprevisíveis têm um impacto negativo no desenvolvimento das plantas. Para responder as estes stresses abióticos e bióticos, as plantas adaptaram-se melhorando os seus complexos mecanismos moleculares, incluindo as modificações pós-tradução (PTMs) que são responsáveis pela modificação de proteínas e consequentemente modelação da sua atividade. Uma importante PTM é a ligação rápida e reversível do péptido SUMO, mais conhecida como sumoilação. Na literatura, está bem descrito que a sumoilação controla inúmeros processos no desenvolvimento da planta e na resposta a stresses. A ligação do SUMO ocorre em quarto passos envolvendo diversos componentes como as ubiquitin-like proteases (ULPs), SUMO ativases (E1), SUMO conjugases (E2) e SUMO ligases (E3). Nesta via, a cascata enzimática E1-E2-E3 é responsável pela ligação do SUMO à proteína alvo. As SUMO proteases ULPs estão envolvidas na maturação do SUMO no primeiro passo da via, mas são também responsáveis pela dessumoilação ou remoção do SUMO da proteína alvo, mantendo o equilíbrio dos níveis de SUMO conjugados. O genoma das plantas codificam para mais ULPs do que os restantes componentes da via de conjugação e, acrescentando à multiplicidade de fenótipos revelados por mutantes das ULPs, estas são consideradas uma fonte de especificidade. Contudo, várias ULP de plantas carecem de caracterização funcional, incluindo a ULP2a e a ULP2b. As primeiras evidências associam a ULP2a e a ULP2b à regulação do desenvolvimento da planta e a inúmeras respostas ao stresse abiótico. Devido à importância destas proteínas, várias estratégias e ferramentas moleculares foram desenvolvidas para estudar a função das ULP2 e encontrar possíveis alvos. No presente trabalho foram cruzadas plantas transgénicas contendo construções SUMO-His com o mutante ulp2a/b para futuros estudos funcionais e purificação/determinação de alvos. Foram produzidas e testadas construções para ensaios de BiFC para visualizar interações com SUMO em plantas. Por fim, proteínas Pds5 ortólogas em plantas, denominadas Pds5-Like Members (PLMs), foram identificadas por via bioinformática como potenciais alvos das ULP2s e aqui usadas como caso de estudo. Plantas mutantes homozigóticas nos genes PLM foram genotipadas, sendo determinados os seus níveis de expressão. Foram de igual forma caracterizadas morfologicamente em condições normais e em resposta ao stresse osmótico. Os genes PLM foram clonadas para futuras purificações de proteína e ensaios de dessumoilação in vitro. O presente trabalho auxiliará o estudo da função das ULP2 e assim ampliará o conhecimento sobre a sumoilação em plantas.<br>Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto “SUMOdulator” (Refs. FCOMP-01-0124-FEDER-028459 e PTDC/BIA-PLA/3850/2012).

碩士<br>國立陽明大學<br>生化暨分子生物研究所<br>100<br>MicroRNAs are endogenous, single-strand RNA molecules that regulate gene expression. MicroRNAs have been identified in different species such as Drosophila, mouse and human. MicroRNA genes located within introns, exons or intergenic areas were transcribed by RNA polymerase II or RNA polymerase III. MicroRNAs are important gene regulators that control cell growth, cell proliferation, cell differentiation and cell death. Recent results have shown that microRNA mutation, amplification, deletion or epigenetic silencing was correlated with various human diseases and cancers. Therefore, microRNA may function as tumor suppressor genes or oncogenes. Epithelial-mesenchymal transition is an important process in cancer metastasis which promote cell migration and cell invasion in cancers. We were interested in identifying novel microRNA(s) involved in epithelial-mesenchymal transition that were induced by hypoxia. From two different predict programs miRBase, TargetScan, the analysis showed that miR-X could target to EMT-relative gene HDAC3 and SENP1. Our preliminary data showed that miR-X could repress luciferase activity of 3’UTR of HDAC3 and SENP1, and the expression of HDAC3 and SENP1 were also repressed in miR-X overexpressing stable lines. miR-X also repress cancer cells migration and invasion ability. For this proposal, I will investigate how hypoxia regulates miR-X expression, and determine how miR-X inhibits the expression of HDAC3 and SENP1. The expression level of miR-X will also be conflated with breast cancer samples to test its prognostic value to predict patients’ survival.

During the activation and transferring process, E1 and E2 form a thioester-linkage with SUMOs. By using an in vitro assay, it is demonstrated that SENP1 is able to cleave the thioester-linkage between SUMO-1/SUMO-3 and E1/E2. This finding suggests that SUMO proteases regulate the sumoylation pathway, not only during maturation and deconjugation, but also in the E1 activation and E2 conjugation processes.<br>Recently, reactive oxygen species have been demonstrated to influence the equilibrium of sumoylation-desumoylation. Here, by in vitro assay, it is shown that H2O2 induces formation of inter-molecular disulfide linkage of human SUMO protease SENP1, via the active-site Cys 603 and a unique residue Cys 613. Such reversible modification confers higher enzyme activity recovery which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. The physiological relevance of the disulfide-linked dimer of SENP1 is also detected in cultured cells upon oxidative stress. The modifications are further verified by the crystal structures of Ulp1 with catalytic cysteine oxidized to sulfenic, sulfinic and sulfonic acids. The current findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors, which modulate the desumoylation pathway and allow immediate specific cellular responses to oxidative stress.<br>SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that is highly conserved in all eukaryotic organisms and regulates cellular function of a variety of target proteins. SUMO proteins are expressed in their precursor forms and precursor processing involves cleavage of the residues after the conserved 'GG' region by the hydrolytic activity of SUMO-specific protease. The exposed second glycine then forms a covalent bond with the epsilon-amino group of a substrate lysine residue at the psiKxE motif by a cascade of SUMO El, E2 and E3 ligases. As a reversible modification, SUMO proteases can cleave SUMOs from their substrates during de-conjugation process.<br>To date, four SUMO family members, SUMO-1, -2, -3 and -4 and six SUMO proteases, SENP1--3 and 5-7 (where SENP stands for sentrin-specific protease) have been identified in human. By characterizing the maturation reactions of SUMO-1, -2 and -3 catalyzed by SENP1, it is demonstrated that SENP1 contains the highest maturation efficiency for SUMO-1, followed by SUMO-2 and SUMO-3. By mutagenesis study, it is further identified that the two amino acids immediately after GG motif could influence the maturation efficiency of SENP1. By comparison with another investigation which showed the preference of the maturation reaction of SUMO-2 by SENP2, the results suggest that SUMO proteases with specific tissue distribution control the availability of different mature SUMOs in human.<br>To gain a deeper insight into the molecular basis of maturation and de-conjugation processes catalyzed by SENP1, it has been determined, at 2.8 A resolution, the X-ray structure of a complex between the catalytic domain of SENP1C(C603S) and matured SUMO-1. The structure shows that the substituted serine residue does not undergo any local structural rearrangements at the active site as observed in the previously solved SENP2/SUMO-1 complex structure. This finding suggests that SUMO proteases require a self-conformational change prior to the cleavage reaction, and further disclose the cleavage mechanism of the hydrolytic reactions catalyzed by SUMO proteases. Moreover, analysis of the interface of SENP1 and SUMO1 has identified four amino acids that are unique in SENP1 sequence and facilitate the interaction of SENP1 and SUMO-1.<br>Xu, Zheng.<br>"July 2007."<br>Advisers: Shannon Au Wing Ngor; Tzi-Bun Ng.<br>Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0125.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.<br>Includes bibliographical references (p. 181-194).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.

Dissertação de mestrado em Molecular Genetics<br>In modern times it has become crucial to provide sustenance for a constantly growing world population, and crop improvement is a current major issue, especially considering the added effects of a changing climate and continuous soil impoverishment. In order to address environmental challenges, plants have developed different defense mechanisms, and studying the way that cells respond to these stresses is an increasingly significant area of research. Post-translational modifications (PTM) are major regulators at the molecular level, increasing the complexity of the cellular proteome. One such PTM is the small ubiquitin-related modifier (SUMO), a ubiquitin-like modifier that was recently shown to have prominent roles in plant development and stress responses. The SUMO pathway consists of three enzymatic steps, where SUMO is first maturated by ubiquitin-like proteases (ULPs), and transferred to the target proteins by SUMO activating (E1) and SUMO conjugating (E2) enzymes, with the assistance of SUMO ligases (E3). The process is recycled by ULPs, which promote deconjugation of the peptide from target proteins. The research group has been involved in the novel characterization of ULP2a and ULP2b SUMO pathway components, using the model plant Arabidopsis thaliana. The current work addressed the functional characterization of these proteins using loss-of-function T-DNA insertion mutants. To characterize the involvement of ULP2s in plant abiotic stress responses, the double mutant ulp2a ulp2b was submitted to a number of different abiotic, hormonal and oxidative stresses. ULP2s displayed roles in osmotic stress, as well as in auxin and ethylene responses. An involvement in heat stress responses was also demonstrated. ULP2 mutants were introgressed into the E3 ligase siz1 mutant, showing that SIZ1 was epistatic to ULP2 in the heat stress response, and acted independently of salicylic acid accumulation in siz1. ULP2- GFP fusion proteins were used to localize ULPs to the cell nucleus. In silico analysis was also performed to predict putative ULP2 targets based on homology from known human and yeast targets. Finally, initial studies were carried out to experimentally establish the gene expression pattern of ULP2s using the GUS reporter system.<br>Nos tempos modernos, tornou-se crucial fornecer o sustento necessário para uma população em constante crescimento, e o melhoramento dos cultivares é um dos principais sectores de preocupação, especialmente considerando os efeitos das alterações climáticas e do contínuo empobrecimento do solo. No sentido de resolver estes desafios ambientais, as plantas desenvolveram diferentes mecanismos defesa, e o estudo da maneira como a célula responde a estes stresses é uma área de investigação de interesse crescente. As modificações pós-traducionais (PTM) são um dos principais mecanismos reguladores a nível molecular, aumentando a complexidade do proteoma celular. Uma destas PTM consiste no pequeno modificador relacionado com a ubiquitina (small ubiquitin-related modifier; SUMO), que se tem demonstrado ter funções importantes no desenvolvimento e na resposta ao stresse em plantas. A via do SUMO consiste em três passos enzimáticos, onde o SUMO é primeiramente maturado pela ação de SUMO proteases (ULPs), e transferido para proteínas alvo pela ação de SUMO ativases (E1) e SUMO conjugases (E2) com a ajuda de SUMO ligases (E3). O ciclo é renovado pelas ULPs, que promovem a desconjugação dos péptidos das proteínas alvo. O grupo de investigação tem estado envolvido na caracterização dos componentes da via do SUMO ULP2a e ULP2b, utilizando como modelo a planta Arabidopsis thaliana. O presente trabalho abordou a caracterização detalhada destas proteínas com a utilização de linhas de inserção de T-DNA. Para caracterizar o envolvimento das ULP2s na reposta a stresses abióticos, o duplo mutante ulp2a ulp2b foi submetido a diferentes stresses abióticos, hormonais e oxidativos. As ULP2s exibiram papéis no stresse osmótico, bem como nas respostas às auxinas e ao etileno. Um envolvimento no stresse pelo calor também foi demonstrado. Os mutantes de ULP2 sofreram introgressão no mutante para a E3 ligase siz1, demonstrando que SIZ1 é epistático para ULP2 na resposta de stresse ao calor, actua de forma independente da acumulação de ácido salicílico em siz1. Fusões ULP2-GFP foram usadas para localizar as ULPs no núcleo da célula. Por sua vez, análises in silico foram efetuadas para prever alvos putativos das ULP2, tendo por base a homologia com alvos de ULP2 em humanos e levedura. Por último, foram iniciados estudos no sentido de estabelecer experimentalmente o padrão de expressão dos genes das ULP2s, utilizando o sistema repórter GUS.

碩士<br>國立臺灣大學<br>生化科技學系<br>102<br>The 26S proteasome is composed of 20S core particle (CP) capped with 19S regulatory particle (RP). Regulatory particle triple-A ATPase 5 (Rpt5) is one of the subunits of the 19S RP, which forms the base of 19S RP together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. The 19S base performs several functions, such as polyubiquitin chain recognition, substrate unfolding, gate opening and also translocation of target proteins into 20S CP. Our previous study has revealed that Rpt5 was modified by small ubiquitin-like modifier 2 (SUMO2) in COS7 cells, and modified by SUMO1 and SUMO2 in the E. coli sumoylation system. In the present study, the in vitro sumoylation assay further demonstrated that recombinant Rpt5 can be modified by SUMO1. Because ATP binding and hydrolysis play critical roles in the regulation of proteasome function, this study was aimed to examine whether SUMO modification may affect the ATPase activity of Rpt5. The result showed that the ATPase activity of Rpt5 was reduced by SUMO2 modification. Furthermore, Rpt5 was found to contain several putative SUMO interacting motifs (SIMs). Although previous in vitro experimental results showed that sumoylation of Rpt5 by SUMO1 was markedly reduced while SIM3 was mutated, the level of Rpt5 sumoylation was not lowered when SIM3 was mutated in HEK293T cells. To rule out the possibility that endogenous Rpt5 might interfere with the observation of sumoylation pattern, shRNAs were applied to knockdown the expression level of endogenous Rpt5. However, the cells with inhibited Rpt5 expression were not viable. Therefore, whether SIM3 is involved in Rpt5 sumoylation remain elusive.

Les estrogènes jouent un rôle primordial dans le développement et le fonctionnement des tissus reproducteurs par leurs interactions avec les récepteurs des estrogènes ERα et ERβ. Ces récepteurs nucléaires agissent comme facteurs de transcription et contrôlent l’expression des gènes de façon hormono-dépendante et indépendante grâce à leurs deux domaines d’activation (AF-1 et AF-2). Une dérégulation de leur activité transcriptionnelle est souvent à l’origine de pathologies telles que le cancer du sein, de l’endomètre et des ovaires. Alors que ERα est utilisé comme facteur pronostic pour l’utilisation d’agents thérapeutiques, l’importance de la valeur clinique de ERβ est encore controversée. Toutefois, des évidences récentes lui associent un pouvoir anti-tumorigénique en démontrant que sa présence favorise l’inhibition de la progression de ces cancers ainsi que l’efficacité des traitements. En combinaisons avec d’autres études, ces observations démontrent que bien que les deux isoformes partagent une certaine similitude d’action, les ERs sont en mesure d’exercer des fonctions distinctes. Ces différences sont fortement attribuables au faible degré d’homologie observé entre certains domaines structuraux des ERs, comme le domaine AF-1, ce qui fait en sorte que les différents sites de modifications post-traductionnelles (MPTs) présents sur les ERs sont très peu conservés entre les isoformes. Or, l’activité transcriptionnelle ligand-dépendante et indépendante des ERs est hautement régulée par les MPTs. Elles sont impliquées à tous les niveaux de l’activation des ERs incluant la liaison et la sensibilité au ligand, la localisation cellulaire, la dimérisation, l’interaction avec l’ADN, le recrutement de corégulateurs transcriptionnels, la stabilité et l’arrêt de la transcription. Ainsi, de par leur dissimilitude, les ERs seront différemment régulés par la signalisation cellulaire. Comme un débalancement de plusieurs voies de signalisation ont été associées à la progression de tumeurs ER-positives ainsi qu’au développement d’une résistance, une meilleure compréhension de l’impact des MPTs sur la régulation spécifique des ERs s’avère essentielle en vue de proposer et/ou développer des traitements adéquats pour les cancers gynécologiques. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre les rôles des MPTs sur l’activité transcriptionnelle de ERβ qui sont, contrairement à ERα, très peu connus. Nous démontrons une régulation dynamique de ERβ par la phosphorylation, l’ubiquitination et la sumoylation. De plus, toutes les MPTs nouvellement découvertes par mes recherches se situent dans l’AF-1 de ERβ et permettent de mieux comprendre le rôle capital joué par ce domaine dans la régulation de l’activité ligand-dépendante et indépendante du récepteur. Dans la première étude, nous observons qu’en réponse aux MAPK, l’AF-1 de ERβ est phosphorylé au niveau de sérines spécifiques et qu’elles jouent un rôle important dans la régulation de l’activité ligand-indépendante de ERβ par la voie ubiquitine-protéasome. En effet, la phosphorylation de ces sérines régule le cycle d’activation-dégradation de ERβ en modulant son ubiquitination, sa mobilité nucléaire et sa stabilité en favorisant le recrutement de l’ubiquitine ligase E6-AP. De plus, ce mécanisme d’action semble être derrière la régulation différentielle de l’activité de ERα et ERβ observée lors de l’inhibition du protéasome. Dans le second papier, nous démontrons que l’activité et la stabilité de ERβ en présence d’estrogène sont étroitement régulées par la sumoylation phosphorylation-dépendante de l’AF-1, processus hautement favorisé par l’action de la kinase GSK-3. La sumoylation de ERβ par SUMO-1 prévient la dégradation du récepteur en entrant en compétition avec l’ubiquitination au niveau du même site accepteur. De plus, contrairement à ERα, SUMO-1 réprime l’activité de ERβ en altérant son interaction avec l’ADN et l’expression de ses gènes cibles dans les cellules de cancers du sein. Également, ces recherches ont permis d’identifier un motif de sumoylation dépendant de la phosphorylation (pSuM) jusqu’à lors inconnu de la communauté scientifique, offrant ainsi un outil supplémentaire à la prédiction de nouveau substrat de la sumoylation. En plus de permettre une meilleure compréhension du rôle des signaux intracellulaires dans la régulation de l’activité transcriptionnelle de ERβ, nos résultats soulignent l’importance des MPTs dans l’induction des différences fonctionnelles observées entre ERα et ERβ et apportent des pistes supplémentaires à la compréhension de leurs rôles physiopathologiques respectifs.<br>Estrogens play a pivotal role in reproductive physiology through direct interaction with the estrogen receptors ERα and ERβ, which belong to the nuclear hormone receptor family of ligand-activated transcription factors. Harbouring two activation domains (AF-1 and AF-2), gene expression can be controlled by ERs either in a hormone-dependent and/or independent manner. Disruption of ER transcriptional regulation is associated with pathological events such as breast and endometrial cancers. While ERα is considered a strong predictive factor in endocrine therapy of reproductive cancers, the clinical value of ERβ is still debated, although greater expression of ERβ has been associated with a favourable outcome since recent evidence has associated ERβ with anti-tumorigenic properties and a better response to anti-estrogenic compounds. Along with others studies, those individual outcomes indicate that even though the two receptors can exert similar roles by sharing resemblances in terms of structure and general response to hormone, they can also carry out distinct functions. These variations can be attributed to the fact that most of the structural domains shared by ERs exhibit a low level of homology, especially at the AF-1 domain. Consequently, the majority of the post-translational modifications sites (PTMs) on ERs are not shared between both isoforms. In fact, ligand-induced and ligand-independent activities of ERs are critically influenced by PTMs. PTMs controls the multiple aspects of ER-dependent activation by modulating ERs ligand binding, specificity, cellular localization, dimerization, interaction with their cognate DNA response element, combinatory recruitment of transcriptional coregulators, stability and transcriptional arrest. Hence, by their discrepancies, ERs will be differently influenced by the cellular environment. Furthermore, as the deregulation of different signalling pathway in cancers is associated with ER-dependant tumour progression and in the acquisition of a therapeutic resistant phenotype, it is crucial to understand the how PTMs affect ERs transactivation in order to eventually propose and/or develop adequate treatment. The results presented in this thesis were carried out with the objective of gaining a better understanding of PTM’s roles on ERβ transcriptional control which, as opposed to ERα, remain unclear. We demonstrate here a dynamic regulation of ERβ by phosphorylation, ubiquitination and sumoylation. Furthermore, as all the newly identified PTM are located within de AF-1 domain of ERβ, our results highlight the key role of this domain in the regulation of ligand-dependent and independent transcriptional properties of this receptor. The first study shows that in response to MAPK, specific serine residues in the AF-1 of ERβ are phosphorylated and play an important role in the regulation of ERβ ligand-independent activity by the ubiquitin-proteasome pathway. In fact, the activation-degradation cycle of ERβ induced by MAPK is regulated upon phosphorylation of these serines coordinating ERβ ubiquitination, subnuclear mobility and stability by promoting the recruitment of the ubiquitin ligase E6-AP. Moreover, this molecular process plays part in the differential regulation of ERα and ERβ activity upon proteasome inhibition. In the second paper, we demonstrate that ERβ activity and stability in presence of estrogen is closely regulated by the phosphorylation-dependent sumoylation of the AF-1 domain, amplified by GSK-3 action. SUMO-1 attachment prevents ERβ degradation by competing with ubiquitin at the same acceptor site and dictates ERβ transcriptional inhibition, as opposed to ERα, by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. Furthermore, these findings uncover a novel phosphorylated sumoylation motif (pSuM) and offer a valuable tool to predict novel SUMO substrates under protein kinase regulation. In combination to our better understanding on how intracellular signals controls ERβ transcriptional activity, our results highlight the significant role of PTMs in ERs isoforms discrepancies and allows supplementary comprehension of their respective physiopathologicals roles.