Letteratura scientifica selezionata sul tema "SUMO Targeted Ubiquitin Ligase"

Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13–Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.

AbstractGordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates.

Candida glabrata is an opportunistic human pathogen, capable of causing severe systemic infections that are often resistant to standard antifungal treatments. To understand the importance of protein SUMOylation in the physiology and pathogenesis of C. glabrata, we earlier identified the components of SUMOylation pathway and demonstrated that the deSUMOylase CgUlp2 is essential for pathogenesis. In this work we show that the CgUlp2 is essential to maintain protein homeostasis via the SUMO-targeted ubiquitin ligase pathway. The dual loss of deSUMOylase and specific ubiquitin ligase, CgSlx8, results in heightened protein degradation, rendering the cells vulnerable to various stressors. This degradation affects crucial processes such as purine biosynthesis and compromises mitochondrial function in the mutants. Importantly, the absence of these ubiquitin ligases impedes the proliferation of C. glabrata in macrophages. These findings underscore the significance of SUMOylation and SUMO-mediated protein homeostasis as pivotal regulators of C. glabrata physiology and capacity to survive in host cells. Understanding these mechanisms could pave the way for the development of effective antifungal treatments.

Small ubiquitin-related modifiers (SUMOs) function as post-translational protein modifications and regulate nearly every aspect of cellular function. While a single ubiquitin protein is expressed across eukaryotic organisms, multiple SUMO paralogues with distinct biomolecular properties have been identified in plants and vertebrates. Five SUMO paralogues have been characterized in humans, with SUMO1, SUMO2 and SUMO3 being the best studied. SUMO2 and SUMO3 share 97% protein sequence homology (and are thus referred to as SUMO2/3) but only 47% homology with SUMO1. To date, thousands of putative sumoylation substrates have been identified thanks to advanced proteomic techniques, but the identification of SUMO1- and SUMO2/3-specific modifications and their unique functions in physiology and pathology are not well understood. The SUMO2/3 paralogues play an important role in proteostasis, converging with ubiquitylation to mediate protein degradation. This function is achieved primarily through SUMO-targeted ubiquitin ligases (STUbLs), which preferentially bind and ubiquitylate poly-SUMO2/3 modified proteins. Effects of the SUMO1 paralogue on protein solubility and aggregation independent of STUbLs and proteasomal degradation have also been reported. Consistent with these functions, sumoylation is implicated in multiple human diseases associated with disturbed proteostasis, and a broad range of pathogenic proteins have been identified as SUMO1 and SUMO2/3 substrates. A better understanding of paralogue-specific functions of SUMO1 and SUMO2/3 in cellular protein quality control may therefore provide novel insights into disease pathogenesis and therapeutic innovation. This review summarizes current understandings of the roles of sumoylation in protein quality control and associated diseases, with a focus on the specific effects of SUMO1 and SUMO2/3 paralogues.

Topoisomerases form transient covalent DNA cleavage complexes to perform their reactions. Topoisomerase I cleavage complexes (TOP1ccs) are trapped by camptothecin and TOP2ccs by etoposide. Proteolysis of the trapped topoisomerase DNA-protein cross-links (TOP-DPCs) is a key step for some pathways to repair these lesions. We describe a pathway that features a prominent role of the small ubiquitin-like modifier (SUMO) modification for both TOP1- and TOP2-DPC repair. Both undergo rapid and sequential SUMO-2/3 and SUMO-1 modifications in human cells. The SUMO ligase PIAS4 is required for these modifications. RNF4, a SUMO-targeted ubiquitin ligase (STUbL), then ubiquitylates the TOP-DPCs for their subsequent degradation by the proteasome. This pathway is conserved in yeast with Siz1 and Slx5-Slx8, the orthologs of human PIAS4 and RNF4.

The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses.

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.

Les défauts dans le processus de réplication de l'ADN, connus sous le nom de stress de réplication, sont une source majeure d'instabilité du génome qui favorise le développement du cancer. La résolution du stress de réplication se produit dans un noyau compartimenté qui présente des capacités distinctes de réparation de l'ADN. Les fourches de réplication stressées présentent une mobilité accrue et se déplacent vers la périphérie du noyau pour s'ancrer aux complexes du pore nucléaire, une structure hautement conservée de l'enveloppe nucléaire qui agit comme un site d'amarrage pour permettre à d'autres voies de réparation de l'ADN de se mettre en place. Ces changements dans le positionnement nucléaire sont régulés par le métabolisme des petits modificateurs de type ubiquitine (SUMO), qui jouent un rôle essentiel dans la ségrégation spatiale des activités de la voie de la recombinaison homologue (RH). Nos travaux antérieurs chez la levure de fission ont établi qu'une fourche de réplication bloquée par une protéine liée à l'ADN se relocalise et s'ancre au NPC d'une manière SUMO-dépendante. Les chaînes SUMO déclenchent la relocalisation des fourches arrêtées à la périphérie du noyau pour s'ancrer au NPC. Cet ancrage nécessite les chaînes SUMO et la voie de l'ubiquitine ligase ciblée par les SUMO (STUbL) Slx8. Cependant, les chaînes Cependant, les chaînes SUMO limitent également la voie de redémarrage de la fourche. Ces conjugués SUMO peuvent être éliminés par la protéase SENP Ulp1 et le protéasome, dont les activités sont enrichies à la périphérie nucléaire. Ainsi, une relocalisation vers les NPCs permet un redémarrage de la réplication dépendant de la RH en contrecarrant la toxicité des chaînes SUMO. La formation de chaînes SUMO et la voie Slx8 étant cruciales pour la relocalisation des fourches de réplications bloquées au NPC. Mon projet s'est d'abord attaché à déterminer si Slx8 STUbL pouvait être exploitée en tant que marqueur des chaînes SUMO induites par des dommages à l’ADN. Pour ce faire, j'ai marqué Slx8 avec une étiquette GFP et j’ai suivi le marquage GFP par microscopie à fluorescence. De manière inattendue, je n'ai pas pu détecter de foyers Slx8 spécifiquement induits par le stress de réplication. Cependant, j'ai découvert que Slx8 forme un foyer nucléaire unique, enrichi à la périphérie nucléaire, qui marque à la fois les centromères groupés au niveau du centre organisateur des microtubules et la région silencieuse du mating type. La formation de ce foyer unique de Slx8 nécessite la ligase E3 SUMO Pli1, la poly-SUMOylation et l'histone méthyl transférase Clr4 qui est responsable de la méthylation de l’histone H3K9 qui marque l'hétérochromatine. Enfin, j'ai établi que Slx8 favorise le regroupement des centromères et le silencing des gènes dans les domaines de l'hétérochromatine. Dans l'ensemble, mes données mettent en évidence des relations fonctionnelles et conservées au cours de l'évolution entre STUbL et les domaines de l'hétérochromatine pour promouvoir le silencing des gènes et l'organisation nucléaire. En outre, j'ai mieux caractérisé les voies de redémarrage des fourches bloquées dans l'espace nucléaire. L'équipe a précédemment établi que les fourches arrêtées nécessitent l'activité d'échange de brins de Rad51 pour être acheminées vers le NPC en vue d'un redémarrage. Dans ce contexte, j'ai dévoilé l'existence d'une voie alternative de redémarrage qui implique la mono-SUMOylation, dans le nucléoplasme en absence de relocalisation au NPC. Ici, je révèle le nouveau rôle de Rad52 dans l'orchestration du redémarrage de la fourche dans le nucléoplasme, un rôle qui implique son activité SSA (single strand annealing). Pris ensemble, mes résultats suggèrent deux aspects. Une partie souligne comment la SUMOylation régulée par Slx8 STUbL favorise la maintenance du centromère. L'autre partie élucide le “contrôle SUMO” des voies alternatives de redémarrage de fourches résolues dans l'espace nucléaire
Flaws in the DNA replication process, known as replication stress, is a major source of genome instability that fuels cancer development. Resolution of replication stress occurs within a compartmentalized nucleus that exhibits distinct DNA repair capacities. In different eukaryotic organisms, stressed replication forks (RFs) shift to the nuclear periphery for anchorage to the nuclear pore complexes (NPCs), a highly conserved structure in the nuclear envelope that act as docking sites to allow alternative DNA repair pathways to occur. These changes in nuclear positioning is regulated by the small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to spatially segregate the activities of the homologous recombination (HR) pathway. Our previous work in the fission yeast Schizossacharomyces pombe, has established that a replication fork blocked by a DNA-bound protein relocates and anchors to NPC in a SUMO-dependent manner. SUMO chains trigger the relocation of single arrested forks to the nuclear periphery to anchor to the NPC. This anchorage requires the SUMO chains and the SUMO-targeted ubiquitin ligase (STUbL), Slx8 pathway. However, SUMO chains also limit the Recombination-Dependent Replication (RDR) pathway, necessary to promote fork restart. These SUMO conjugates can be cleared off by the SENP protease Ulp1 and the proteasome, whose activities are enriched at the nuclear periphery. Thus, a routing towards NPCs allows HR-dependent replication restart by counteracting the toxicity of SUMO chains. Since, both SUMO chain formation and the Slx8 STUbL pathway were crucial for NPC routing of arrested replication forks. My thesis project initially focused on unraveling if the Slx8 STUbL can be exploited as a readout of damage-induced SUMO chains. To do so, I tagged Slx8 with a GFP tag and monitored them using the fluorescence microscopy technique. Unexpectedly, I was unable to detect replication stress-induced Slx8 foci. However, I discovered that Slx8 forms a single nuclear focus, enriched at the nuclear periphery, which marks both clustered centromeres at the spindle pole body and the silent mating type region. The formation of this single Slx8 focus requires the E3 SUMO ligase Pli1, poly-SUMOylation and the histone methyl transferase Clr4 that is responsible for the heterochromatin histone mark H3-K9 methylation. Finally, it was established that Slx8 promotes centromere clustering and gene silencing at heterochromatin domains. Altogether, my data highlight evolutionarily conserved and functional relationships between STUbL and heterochromatin domains to promote gene silencing and nuclear organization. Additionally, I have better characterized pathways of fork restart within the nuclear space. The team previously established that arrested RFs require SUMO chains and the strand exchange activity of Rad51 for routing to the NPC for subsequent fork restart. In this context, I unveiled the existence of an alternate fork restart pathway that occurs by mono-SUMOylation, in the nucleoplasm when forks do not shift to the NPC, as SUMO chains are not formed. Here, I revealed that fork restart within the nucleoplasm still depends on the strand exchange activity of Rad51 largely, while the single strand annealing (SSA) activity of Rad52 plays an important role in mediating error-prone fork progression in the absence of SUMO chains. Taken together, my results suggest two different ideas about SUMOylation. One part underscores how Slx8 STUbL-regulated SUMOylation promotes centromere clustering and gene silencing at heterochromatin domains. Whereas, the other section elucidates the “SUMO control” on the spatially segregated, alternative pathways of fork restart within the nuclear space. Therefore highlighting the importance of maintaining SUMO balance for preserving genome integrity

La réparation de cassures d’ADN double brins par recombinaison homologue nécessite la formation d’intermédiaires multibrins qui peuvent être le lieu de formation de crossovers après résolution par des nucléases. La modification de protéines par ubiquitine et SUMO est un mode de contrôle répandu parmi les protéines de la réparation de l’ADN. De plus, certaines protéines de la réparation de cassures double brins interagissent entre elles, lorsqu’elles sont sumoylées, par le biais de motifs d’interaction avec SUMO (SIMs). La nucléase Yen1 subit un contrôle rigoureux lors du cycle cellulaire dans le but de limiter la formation de crossover et ainsi de préserver l’intégrité du génome. Dans ce manuscrit, il sera mis en évidence que Yen1 est régulé de surcroit par l’ubiquitination, la sumoylation et enfin l’interaction non covalente avec le modificateur SUMO via ses SIMs désormais découverts. Yen1 est sumoylé par les SUMO ligases Siz1 et Siz2, d’autant plus en conditions de dommages à l’ADN. En plus de quoi, Yen1 est un substrat de l’ubiquitine ligase Slx5-Slx8. En absence de cette dernière, la fraction sumoylée de Yen1 persiste, ce qui mène à la localisation durable de Yen1 en accumulation ponctuelle dans le noyau. L’ubiquitination de Yen1 par Slx5-Slx8 a surtout lieu à la lysine 714. Une mutation de cette lysine augmente la formation de crossovers, et annule également les défauts de ségrégation des chromosomes qui peuvent avoir lieu en l’absence d’autres nucléases. D’autre part, l’action nucléolytique de Yen1 ne s’effectue correctement que lorsque celui-ci peut interagir de façon non covalente avec des partenaires sumoylés. Des mutations dans les SIMs de Yen1 réduisent sa capacité à découper et résoudre les intermédiaires de la recombinaison, ce qui donne lieu à une augmentation de l’instabilité génomique et de la mauvaise ségrégation des chromosomes
The repair of double-stranded DNA breaks (DSBs) by homologous recombination involves the formation of branched intermediates that can lead to crossovers following nucleolytic resolution. Ubiquitin and SUMO modification is commonplace amongst the DNA damage repair proteins. What is more, a number of DSB repair factors interact with each other when sumoylated, making use of SUMO interaction motifs (SIMs). The nuclease Yen1 is tightly controlled during the cell cycle to limit the extent of crossover formation and preserve genome integrity. In this manuscript we describe further regulation of Yen1 by ubiquitination, sumoylation and non-covalent interaction with SUMO through its newly characterized SIMs. Yen1 is sumoylated by Siz1 and Siz2 SUMO ligases, especially in conditions of DNA damage. Furthermore, Yen1 is a substrate of the Slx5-Slx8 ubiquitin ligase. Loss of Slx5-Slx8 stabilizes the sumoylated fraction of Yen1, and results in persistent localization of Yen1 in nuclear foci. Slx5-Slx8-dependent ubiquitination of Yen1 occurs mainly at K714 and mutation of this lysine increases crossover formation during DSB repair and suppresses chromosome segregation defects when other nucleases are unavailable. In addition, proper and timely nucleolytic processing from Yen1 is dependent on interactions mediated by non-covalent binding to sumoylated partners. Mutations in the motifs that allow SUMO-mediated recruitment of Yen1 leads to its mis-localization, decreasing Yen1’s ability to resolve DNA joint-molecule intermediates and resulting in increased genome instability and chromosome mis-segregation

Previously it has been shown that Rsp5p, a member of Nedd4 ubiquitin ligases in yeast, is modified by the ubiquitin-like protein SUMO and that this modification is performed by Siz1p, a member of PIAS SUMO ligases that are in turn substrates of Rsp5p-dependent ubiquitylation, thus defining a previously unidentified system of crosstalk between the ubiquitin and SUMO systems in yeast. This project aims to identify whether similar crosstalk pattern exists in human cells. In vitro ubiquitylation assays showed that some of the human Nedd4 family members (Nedd4.1, Nedd4.2, WWP1) are capable of ubiquitylating the human SUMO ligase PIAS3, while in contrast, Smurf2 does not appear to be able to modify this protein. This modification is partially WW-PY-motif-dependent as ubiquitylation level of PIAS3 mutants with altered PY motifs conducted by Nedd4.1 or Nedd4.2 was reduced, but not completely disrupted. Interestingly, in vitro SUMOylation assay revealed that Nedd4.1 is SUMOylated even in the absence of SUMO E3 ligases and an apparent interaction between the SUMO E2 (Ubc9) and Nedd4.1 was observed both in vitro and in vivo. I show that auto- SUMOylation of Nedd4.1 is accompanied with the formation of thioester-linked conjugates between Nedd4.1 and SUMO, but these do not involve cysteine residues (C867, C778, and C627) within the HECT domain itself and is not occurring at a predicted SUMOylation consensus site (K357). Furthermore, I have shown that Nedd4.1 and SUMO1/2 colocalize in HeLa cells, and that overexpression of epitope tagged Nedd4 and SUMO1/2, followed by denaturing pull-downs demonstrates that both Nedd4.1 and Nedd4.2 can be SUMOylated in vivo. Meanwhile, I have generated a SUMO trap based on SUMO interacting motifs (SIMs) and confirmed its ability of capturing SUMOylated proteins both in vivo and in vitro. Its use reveals that Nedd4 SUMO conjugates could be captured by SUMO trap when Nedd4 and SUMO were co-expressed in HeLa cells, again confirming Nedd4.1 as a substrate for SUMO1 or SUMO2. In conclusion, I show that SUMOylation of Nedd4.1 does exist in HeLa cells, and on the other hand, some of Nedd4 family members are responsible for PIAS3 ubiquitylation in vitro, providing evidence of a crosstalk between Nedd4 family of ubiquitin ligases and PIAS family of SUMO ligases in mammals.

La modification des protéines par SUMO joue un rôle important dans de nombreuses fonctions cellulaires. Le travail présenté dans cette thèse vise à analyser l'action des protéines PIAS, régulateurs transcriptionnels et SUMO E3 ligases, et en particulier de PIASy dans le contexte de l'oncogenèse. Nous avons montré que PIASy induit la sénescence des fibroblastes primaires, en activant les voies Rb et p53, ou l'apoptose si la voie Rb est déficiente. Nous avons parallèlement identifié de nouveaux partenaires protéiques de PIASy. PIASy induit l'accumulation de FIP200 dans le noyau, ce qui inhibe l'action de FIP200 dans la voie mTOR. FIP200 coopère avec PIASy pour activer l'expression du gène p21. Par ailleurs, la modification de PARP-1 par SUMO stimulée par PIASy régule la réponse au choc thermique. Ainsi, ce travail a mis en évidence un rôle important de PIASy dans le contrôle de la prolifération cellulaire et la réponse aux dommages, et a précisé son action au niveau moléculaire

Trois protéines de la famille TRIM (Motif TRIpartite), TIF1α, β (Transcriptional Intermediary Factor 1) et PML (ProMyelocytic Leukaemia¬), font l’objet de cette étude. TIF1α est connu comme un coactivateur des récepteurs nucléaires et TIF1β comme le corépresseur universel des protéines KRAB-multidoigt de zinc dont le prototype étudié ici est ZNF74. PML possède divers rôles dont le plus caractérisé est celui d’être l’organisateur principal et essentiel des PML-NBs (PML-Nuclear Bodies), des macrostructures nucléaires très dynamiques regroupant et coordonnant plus de 40 protéines. Il est à noter que la fonction de TIF1α, β et PML est régulée par une modification post-traductionnelle, la sumoylation, qui implique le couplage covalent de la petite protéine SUMO (Small Ubiquitin like MOdifier) à des lysines de ces trois protéines cibles. Cette thèse propose de développer des méthodes utilisant le BRET (Bioluminescence Resonance Energy Transfert) afin de détecter dans des cellules vivantes et en temps réel des interactions non-covalentes de protéines nucléaires mais aussi leur couplage covalent à SUMO. En effet, le BRET n’a jamais été exploré jusqu’alors pour étudier les interactions non-covalentes et covalentes de protéines nucléaires. L’étude de l’interaction de protéines transcriptionnellement actives est parfois difficile par des méthodes classiques du fait de leur grande propension à agréger (famille TRIM) ou de leur association à la matrice nucléaire (ZNF74). L’homo et l’hétérodimérisation de TIF1α, β ainsi que leur interaction avec ZNF74 sont ici testées sur des protéines entières dans des cellules vivantes de mammifères répondant aux résultats conflictuels de la littérature et démontrant que le BRET peut être avantageusement utilisé comme alternative aux essais plus classiques basés sur la transcription. Du fait de l’hétérodimérisation confirmée de TIF1α et β, le premier article présenté ouvre la possibilité d’une relation étroite entre les récepteurs nucléaires et les protéines KRAB- multidoigt de zinc. Des études précédentes ont démontré que la sumoylation de PML est impliquée dans sa dégradation induite par l’As2O3 et dépendante de RNF4, une E3 ubiquitine ligase ayant pour substrat des chaînes de SUMO (polySUMO). Dans le second article, grâce au développement d’une nouvelle application du BRET pour la détection d’interactions covalentes et non-covalentes avec SUMO (BRETSUMO), nous établissons un nouveau lien entre la sumoylation de PML et sa dégradation. Nous confirmons que le recrutement de RNF4 dépend de SUMO mais démontrons également l’implication du SBD (Sumo Binding Domain) de PML dans sa dégradation induite par l’As2O3 et/ou RNF4. De plus, nous démontrons que des sérines, au sein du SBD de PML, qui sont connues comme des cibles de phosphorylation par la voie de la kinase CK2, régulent les interactions non-covalentes de ce SBD mettant en évidence, pour la première fois, que les interactions avec un SBD peuvent dépendre d’un évènement de phosphorylation (“SBD phospho-switch”). Nos résultats nous amènent à proposer l’hypothèse que le recrutement de PML sumoylé au niveau des PML-NBs via son SBD, favorise le recrutement d’une autre activité E3 ubiquitine ligase, outre celle de RNF4, PML étant lui-même un potentiel candidat. Ceci suggère l’existence d’une nouvelle relation dynamique entre phosphorylation, sumoylation et ubiquitination de PML. Finalement, il est suggéré que PML est dégradé par deux voies différentes dépendantes de l’ubiquitine et du protéasome; la voie de CK2 et la voie de RNF4. Enfin une étude sur la sumoylation de TIF1β est également présentée en annexe. Cette étude caractérise les 6 lysines cibles de SUMO sur TIF1β et démontre que la sumoylation est nécessaire à l’activité répressive de TIF1β mais n’est pas impliquée dans son homodimérisation ou son interaction avec la boîte KRAB. La sumoylation est cependant nécessaire au recrutement d’histones déacétylases, dépendante de son homodimérisation et de l’intégrité du domaine PHD. Alors que l’on ne connaît pas de régulateur physiologique de la sumoylation outre les enzymes directement impliquées dans la machinerie de sumoylation, nous mettons en évidence que la sumoylation de TIF1β est positivement régulée par son interaction avec le domaine KRAB et suggérons que ces facteurs transcriptionnels recrutent TIF1β à l’ADN au niveau de promoteur et augmentent son activité répressive en favorisant sa sumoylation.
Three TRIM proteins (TRIpartite Motif), TIF1α, β (Transcriptional Intermediary Factor 1) and PML (ProMyelocytic Leukaemia¬), were studied in this thesis. TIF1α is a nuclear receptor coactivator and TIF1β is the universal corepressor of the KRAB-zinc finger repressor family of which, ZNF74 is studied here as a prototypic member. PML functions as a tumor suppressor and is the essential organiser of PML-NBs (PML-Nuclear Bodies) which are very dynamic nuclear macrostructures containing more than 40 proteins. The function of these three TRIM proteins is regulated by sumoylation, a post-translational modification involving the covalent linkage of SUMO (Small Ubiquitin like MOdifier) to specific targets lysine. In this thesis, we propose to develop new methods based on BRET (Bioluminescence Resonance Energy Transfer) to detect non-covalent nuclear protein interactions but also covalent linkage to SUMO in real time in living cells. To date, BRET was never used to assess non-covalent or covalent nuclear protein interactions. Studying transcriptionally active protein interactions represents a challenge by classical methods in particular when proteins have a tendency to aggregate (TRIM family) or when characterizing nuclear matrix proteins (ZNF74). In the first article, homo- and heterodimerisation of TIF1 α and β as well as their interaction with ZNF74 was assessed by BRET using full length proteins in living mammalian cells. We ascertained the heterodimerisation of TIF1α and β. Whereas ZNF74 interacts strongly with TIF1β, no interaction was detected with TIF1α. However, we unravelled the existence of ternary complexes involving ZNF74, TIF1α and TIF1β. This suggested that a mechanisms for cross-talk between nuclear receptors and KRAB-zinc finger proteins. Thus, we showed that BRET can be advantageously used as a non-transcription-based interaction system for studying transcriptionally active proteins, including nuclear matrix proteins, in living cells. Previous studies have shown that the sumoylation of PML (a tumour suppressor) is involved in its proteasome degradation that is As2O3-inducible and dependent on the polySUMO E3 ubiquitin ligase, RNF4. In the second article, we describe the development of a new application of the BRET method for the detection of covalent and non-covalent interactions with SUMO. Owing to this SUMO BRET assay, we established that the As2O3 / RNF4-mediated degradation of PML, not only depends on PML sumoylation as previously demonstrated, but also on the integrity of its SUMO binding domain. We also demonstrated that As2O3 which increases PML sumoylation, also enhances PML / RNF4 interaction. Our study revealed that most PML SBD non covalent interactions with sumoylated proteins required the phosphorylation of serines within PML SBD that were previously described as target sites for CK2 kinase and involved in PML degradation. Despites the involvement of PML SBD in RNF4-mediated degradation, these serines which function as an SBD phospho-switch, were not required for RNF4-mediated degradation. This suggested that CK2- and RNF4-mediated PML degradation represents two distinct pathways triggering PML ubiquitin / proteasome-dependent degradation. At last, our study led to the hypothesis that the recruitment of sumoylated PML at PML-Nuclear Bodies subnuclear structures via the PML SBD and / or possibly an E3 ubiquitin ligase activity other than RNF4 (PML itself being candidate) may favour PML degradation. Our study also stresses the dynamic involvement of three PML post-translational modifications, phosphorylation, sumoylation and ubiquitination in its degradation. A third article addressing the role of TIF1β sumoylation is presented in the Appendix. We characterized the 6 SUMO targets lysine of TIF1β and demonstrated that sumoylation is required for TIF1β transcriptional repressive activity. This is in part explained by the fact that TIF1β sumoylation is a pre-requisite for histone deacetylases recruitment since TIF1β repressive activity is partly dependent on histone deacetylases. We found that TIF1β sumoylation does not influence its homodimerisation or interaction with the KRAB box of KRAB zinc finger proteins recruiting TIF1β to promoters. TIF1β sumoylation is however relying on the integrity of TIF1β PHD finger and on its self-oligomerisation. Interestingly, we demonstrated that TIF1β sumoylation is positively regulated by its interaction with KRAB domain. It is thus suggested that KRAB-zinc finger proteins recruit TIF1β at DNA promoters where they trigger increase of TIF1β sumoylation and thus enhance its repressive activity.

Abstract Recent experimental evidence implicates the ubiquitin system in the specific and programmed degradation of many key regulatory and short-lived cellular proteins. Among these are mitotic cyclins, transcriptional activators, tumour suppressors, and growth regulators, as well as membrane receptors involved in signal transduction and the immune and inflammatory responses. It is also involved in processing of antigens for presentation via class I MHC molecules. The system can specifically target abnormal, misfolded, and unassembled proteins that may result from mutations, environmental damage, or non stoichiometric synthesis of subunits of complex proteins. It is clear therefore that ubiquitin-mediated proteolysis plays important roles in the regulation of basic pathophysiological cellular processes such as regulation of cell cycle and division, development and differentiation, malignant transformation, modulation of the immune system, and neurodegeneration. With the multitude of substrates targeted, it is not surprising that recent studies have implicated the system in the pathogenesis of several disease states as well. For example, the inability to degrade the kidney Na+ channel leads to stabilization of the protein, accumulation of the channel subunits, excessive reabsorption of the cation and water, and a severe form of hypertension (1). Mutations in the gene that encodes E6-AP, a ubiquitin-protein substrate ligase, lead to the Angelman syndrome that is characterized by mental retardation, seizures, and abnormal gait (2). It is predicted that the list of ubiquitin-related pathological derangements will grow significantly in the future.