L’incidence du mélanome a augmenté de façon considérable lors des trente dernières années avec un doublement tous les dix ans. Le mélanome ne représente que 5% des cancers cutanés mais entraîne 80% de décès, ce qui constitue un problème majeur de santé publique. En effet, cette tumeur est extrêmement agressive et possède un fort potentiel métastatique. Dès l’apparition de métastases, le pronostic vital devient fortement défavorable. Malgré des avancées thérapeutiques majeures, de nombreux patients sont encore réfractaires à ces nouveaux traitements. La compréhension des mécanismes impliqués dans le développement de cette tumeur reste donc un enjeu de premier ordre. Le séquençage d'exomes a conduit à l'identification d'une mutation dans le gène RAC1 (la mutation P29S) constituant une des mutations somatiques les plus fréquentes dans le mélanome (après les mutations BRAFV600, NRASQ61 et NF1). RAC1 est une petite GTPase qui fonctionne dans plusieurs processus cellulaires. Dans des conditions physiologiques, l'activité de RAC1 est principalement contrôlée par des protéines activatrices de l'activité GTPase (GAPs) et des facteurs d'échange Nucléotidique (GEF). GAPs et GEFs contrôlent le niveau de RAC1-GTP et régulent donc son activité. L'activité de RAC1 est aussi dépendante de son niveau d'expression protéique qui est contrôlé par des E3 ubiquitine ligases, parmi lesquelles HACE1. HACE1 est considérée comme un suppresseur de tumeur. De façon inattendue, les données obtenues montrent clairement que HACE1 favorise les propriétés migratoires et tumorigéniques des cellules de mélanome. / Melanoma incidence has considerably increased over the last thirty years, with a doubling every ten years. Melanoma accounts for only 5% of cutaneous cancers but causes more than 80% of deaths, which is a major public health problem. Indeed, this tumor is extremely aggressive and has a high metastatic potential. After the onset of metastases, the prognosis becomes highly unfavorable. Despite major therapeutic advances, many patients are still refractory to these new treatments. Understanding the mechanisms involved in the development of this tumor and the identification of new therapies remain a major issue. The sequencing of exomes led to the identification of a mutation in the RAC1 gene (P29S) constituting one of the most frequent somatic mutations in melanoma (after the BRAFV600, NRASQ61 and NF1 mutations). RAC1 is a small GTPase that is involved in several key cellular processes. Under physiological conditions, the activity of RAC1 is mainly controlled by GTPase activating proteins (GAPs) and Nucleotide Exchange (GEF) exchange factors. GAPs and GEFs control the level of RAC1- GTP and thus regulate its activity. The activity of RAC1 is also dependent on its protein level of expression which is controlled by E3 ubiquitin ligases, including HACE1. HACE1 is considered a tumor suppressor. Unexpectedly, our data clearly show that HACE1 promotes migratory and tumorigenic properties of melanoma cells. Indeed, inhibition of HACE1 alters migration of melanoma cells in vitro, as well as in vivo pulmonary colonization in mice. Transcriptomic analysis of 4 melanoma cell lines demonstrated that HACE1 suppression inhibits ITGAV and ITGB1 expression.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

Camptothecin and its analogues (CPTs) represent one of the most potent classes of anticancer drugs used to treat several solid tumors. CPTs bind topoisomerase I during the replication process and cause DNA damage that results in cell death. However, its effectiveness is limited to 13-30 percent of patients. TopoI cuts and re-ligates DNA supercoiling but in the presence of CPT it fails to re-ligate DNA and collision of replication forks leads to DNA double strand break (DNA-DSB) and cell death. However, in resistant cells, due to deregulated kinase cascade, topoI is continually phosphorylated by DNA-PKcs and rapidly degraded by the ubiquitin proteasomal pathway (UPP). It has been found that BRCA1 plays a key role in imparting cellular resistance to topoI inhibitors. Importantly, BRCA1 ubiquitinates topoI in response to CPT. We hypothesize that disruption of BRCA1 binding to phosphorylated topoI would interrupt the resistance mechanism resulting in higher cellular sensitivity of CPT. Based on an in-silico drug screen, we identified a compound that inhibits topoI degradation by blocking BRCA1 binding. Imaging and survival assays findings are consistent with the hypothesis that BRCA1 plays a role in CPT resistance through its co-localization with topoI, and we speculate this role is through UPP degradation. CPTs are commonly used in combination with cytotoxic compounds, but this study focuses on discovering compounds that can overcome resistance without causing further cytotoxicity. / 2019-07-03T00:00:00Z

Changes in cellular redox status are implicated in the regulation of developmental and defence-related responses. The absence of S-nitrosoglutathione reductase (GSNOR) function in Arabidopsis leads to an accumulation of cellular S-nitrosoglutathione (GSNO), a mobile reservoir of nitric oxide (NO) which impacts the cellular redox tone. Consequently, the GSNOR knockout mutant, atgsnor1-3 displays defects in growth, time to flowering and pathogen resistance. Although it is now well established that GSNO is a key redox signalling molecule, the molecular mechanisms that underpin GSNO function remains largely unknown. RAP1 (REDOX-ASSOCIATED PROTEIN 1) was identified based on its dynamic changes of expression in atgsnor1-3 and sid2 plants upon avirulent Pseudomonas syringae pv. tomato (Pst) DC3000 (avrB) challenge. Pathogen-induced RAP1 expression was shown to be independent of the plant hormones salicylic acid, jasmonic acid, abscisic acid and ethylene. Recombinant RAP1 protein was shown to exhibit E3 ligase activity in vitro. Application of the NO donors (GSNO and Cysteine-NO (CysNO)) reduced the E3 ligase activity of RAP1 significantly. Biotinswitch analysis showed that RAP1 was S-nitrosylated and site-directed mutagenesis of RAP1 suggested that the S-nitrosylated site is the cysteine residue C325. The rap1 line does not show obvious developmental phenotypes, however, overexpressing RAP1 enhanced lateral root branching in young seedlings. Overexpression of a truncated RAP1 (RAP1ΔRING) led to a loss of apical dominance. In addition, rap1/rap2 double mutants showed delayed flowering, suggesting RAP1 might be involved in the regulation of plant growth and development. RAP1 may also be involved in plant defence, as rap1, rap2 and rap1/rap2 mutants exhibited increased susceptibility to PstDC3000 and Arabidopsis powdery mildew. Interestingly, rap1 plants showed enhanced resistance to methyl viologen (MV), which is in line with the phenotype of atgsnor mutants. Also, expression of RAP1 was rapidly inducible by ultraviolet-B (UV-B) light. As RAP1 expression and RAP1 E3 ligase activity are redox-related, it is speculated that RAP1 may be involved in redoxmediated regulation of a broad range of physiological responses.

The BCA2 E3 ligase is expressed in a majority of invasive breast cancers. BCA2 has inherent autoubiquitination activity which contributes to cell migration and proliferation processes. Here, ten novel BCA2 binding proteins were found using yeast and bacterial screening. Two of which were human homolog of Rad23 variant A (hHR23a) and 14-3-3σ. In vivo and in vitro assays confirmed that both hHR23a and 14-3-3σ bound BCA2 and were co-expressed with BCA2 in breast cancer cells. Interaction of BCA2 with hHR23a and 14-3-3σ affect the autoubiquitination and auto-degradation activity of BCA2. Multi-ubiquitination of hHR23a-bound BCA2 was dramatically lower than that of free BCA2, this corresponded to increased BCA2 expression and half-life. Furthermore, phosphorylated BCA2 protein was stabilized by interaction with 14-3-3σ, via substrate inhibition of BCA2 autoubiquitination. High expression of BCA2 is correlated with grade in breast cancer and regulation of this E3 ligase’s activity may be important to cancer progression.

The BCA2 E3 ligase is expressed in a majority of invasive breast cancers. BCA2 has inherent autoubiquitination activity which contributes to cell migration and proliferation processes. Here, ten novel BCA2 binding proteins were found using yeast and bacterial screening. Two of which were human homolog of Rad23 variant A (hHR23a) and 14-3-3σ. In vivo and in vitro assays confirmed that both hHR23a and 14-3-3σ bound BCA2 and were co-expressed with BCA2 in breast cancer cells. Interaction of BCA2 with hHR23a and 14-3-3σ affect the autoubiquitination and auto-degradation activity of BCA2. Multi-ubiquitination of hHR23a-bound BCA2 was dramatically lower than that of free BCA2, this corresponded to increased BCA2 expression and half-life. Furthermore, phosphorylated BCA2 protein was stabilized by interaction with 14-3-3σ, via substrate inhibition of BCA2 autoubiquitination. High expression of BCA2 is correlated with grade in breast cancer and regulation of this E3 ligase’s activity may be important to cancer progression.

Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.

Estudos preliminares através de microarray nos mostraram que a E3 ligase Mdm2 foi regulado positivamente no músculo de animais hipertireoideos. Dessa forma, nós inferimos uma possível relação de Mdm2 com a atrofia causada por T3. Para testar nossa hipótese, ratos foram induzidos ao hipertireoidismo para análises subsequentes. Concomitante com a perda de massa muscular foi confirmado um aumento da expressão de Mdm2 tanto no nível gênico (p<0.05) quanto protéico. Interessantemente, Mdm2 foi preferencialmente expresso em fibras tipo I, mostrando maior sensibilidade dessas fibras ao T3. Além disso, foi observado uma diminuição severa na expressão de Pax7/MyoD associado à superexpressão de Mdm2, sugerindo inatividade das células satélites. Surpreendentemente, a inibição de Mdm2 em miotubos cultivados provocou uma diminuição severa no diâmetro destes (~35%, p<0.05), ou seja, tal inibição foi incapaz de minimizar a proteólise muscular causada por T3. Portanto, nós concluímos que a responsividade de Mdm2 ao T3 agiria como um mecanismo compensatório numa tentativa de minimizar a proteólise muscular causada pelo hipertireoidismo. Esta conclusão é reforçada pela atrofia observada em miotubos durante a inibição de Mdm2 sem a presença de T3. / Previous studies in our lab through microarray assay observed Mdm2, an E3 ligase, up regulated in soleus muscle from hyperthyroid rats. In this sense, we inferred that Mdm2 could be related to muscle atrophy caused by T3. To test our hypothesis, rats were induced to experimental hyperthyroidism for subsequent analysis. Along the muscle mass loss, the increase on Mdm2 gene expression was confirmed (p<0.05) as well as protein expression by RT-PCR and Western Blot, respectively. Interestingly, Mdm2 was expressed predominantly in fiber I type during T3 treatment, demonstrating a higher sensibility when compared to type II fiber. Moreover, it was observed a severe decrease in Pax7/MyoD labeling, associated to an increase on Mdm2 labeling, suggesting that T3 could be associated with inactivation of satellite cells. Surprisingly, Mdm2 inhibition in myotubes have induced severe decrease on myotubes diameter (~35%, p<0.05), in other words, Mdm2 inhibition was not able to decrease muscle proteolysis during high levels of T3. Thus, the increase on Mdm2 levels could be a compensatory effect to reduce the muscle mass loss during T3 treatment. This conclusion is highlighted by the myotubes atrophy observed during the Mdm2 inhibition without T3 treatment.

Le blé tendre, Triticum aestivum, représente une grande ressource dans l’alimentation humaine mais également dans l’industrie. En conséquence, la taille finale du grain de blé constitue une cible privilégiée des programmes de sélections variétales. Pour ces raisons, comprendre les mécanismes moléculaires qui contrôlent le développement du grain, en particulier lors des phases précoces de la mise en place des structures cellulaires et leur remplissage par des réserves, constitue un enjeu majeur. Le développement du grain de blé est un processus complexe qui nécessite l’intervention séquentielle ou combinée d’un très grand nombre de gènes et de voies métaboliques. Parmi ces voies, le métabolisme carboné (en particulier celui du saccharose), les voies de signalisation par les hormones et la voie Ubiquitine / Protéasome 26S (UPS) semblent jouer un rôle déterminant dans la taille finale et donc le rendement en grain chez les céréales. Pour étudier le développement du grain de blé tendre, des plantes de la variété Récital ont été cultivées en serre dans des conditions optimales sans contraintes. Les grains ont été récoltés à onze stades de développement après floraison, allant de 40°CJ (soit deux jours après floraison) à 500°CJ (soit 25 jours après floraison). Les ARN totaux ont été extraits à partir de ces grains et utilisés pour analyser l’expression des gènes par une approche transcriptomique, soit en utilisant une lame « dédiée », soit en utilisant une lame Nimblegen comprenant 39 179 gènes. Une analyse différentielle utilisant le test LIMMA a permis d’identifier 9284 gènes différentiellement exprimés. L’analyse globale de ces gènes a montré que des modifications transcriptionnelles majeures ont lieu entre les stades 80 et 120 °CJ ainsi qu’entre 220 et 240°CJ. La répartition de ces 9284 en 10 clusters, en fonction de leurs profils d’expression, permet d’identifier les gènes activés en début de la phase de division cellulaire chez le grain, ceux activés pendant la phase de remplissage et ceux présentant un profil dit en « cloche ». Parmi les gènes différentiellement exprimés, nous nous sommes intéressés à ceux qui codent pour des E3 ligases impliquées dans la voie UPS et aux gènes relatifs à 7 hormones végétales (auxine, acide abscissique, acide jasmonique, brassinostéroïdes, cytokinines, gibbérellines et éthylène). Nous avons alors identifié 173 gènes codant pour des E3 ligases (dont certaines sont également des récepteurs hormonaux) et 126 gènes impliqués dans les voies hormonales. Un modèle global décrivant la chronologie d’intervention de ces gènes a été proposé. La majorité des gènes E3 de type SCF (SKP1-Cullin-Fbox), APC/C, Cul3-BTB et Ubox interviendrait dans les phases précoces du développement du grain de blé. Parallèlement, la majorité des gènes relatifs à l’auxine, à l’acide jasmonique et aux brassinostéroïdes interviendrait lors des phases de divisions cellulaires alors que les gènes relatifs à l’éthylène et à l’acide abscissique interviendraient dans les phases de remplissage. Par ailleurs, une méta-analyse a été réalisée et a permis d’identifier 26 gènes candidats codants pour des E3 ligases ainsi que de 12 gènes candidats impliqués dans les voies hormonales qui seraient préférentiellement exprimés dans l’albumen, un tissu du grain à haute valeur agro-économique. Le modèle proposé et l’identification de ces gènes candidats établissent un cadre pour de futures études visant à comprendre les mécanismes moléculaires contrôlant le développement du grain de blé. / Wheat grain is an important source of food, feed, and industrial raw materials, but current production levels cannot meet world needs. Elucidation of the molecular mechanisms underlying wheat grain development will contribute valuable information to improving wheat cultivation. One of the most important mechanisms implicated in plant developmental processes is the Ubiquitin-Proteasome System (UPS). Among several implications of the UPS, it has become clear that it plays an essential role in hormone signaling. In particular E3 ubiquitin ligases, from the UPS, have been demonstrated to play critical roles in hormone perception and signal transduction. During these work, wheat cv. Recital were grown in optimum growth conditions. By comparing eleven consecutive time-points from 40°CJ (2 days after anthesis) to 500°CJ (around 25 days after anthesis), 9284 differentially expressed genes were identified during this study. A comparison of these genes in terms of time revealed dynamic transcript accumulation profiles with major re-programming events that occurred during the time intervals of 80-120°Cdays and 220-240°Cdays. The gene expression comparison allows observing genes potentially involved in cell division or grain filling stage. An emphasis was made on the E3 ligases and hormone-related genes (Abscisic acid, Auxin, Brassinosteroid, Cytokinine, Gibberellic acid, Ethylene and Jasmonic acid). 173 E3 ligase coding genes and 126 hormone–related genes were found to be differentially expressed during the cell division and grain filling stages, with a different expression profile for each family. A model describing the timing of the involvement of these genes is proposed to provide a framework for the design of future experiments and for the identification of genes and pathways for further characterization. A majority of the E3 SCF (SKP1-Cullin-F-box), APC/C, Cul3-BTB and Ubox are found expressed in early wheat developmental stages (cell division stage). A majority of auxin, jasmonic acid and brassinostéroïde related genes were found to be up-regulated in early wheat developmental stages while ethylene and abscisic acid related genes were found to be activated during grain filling stage. The differential expression of genes involved in E3 ligase pathways and plant hormone signalling suggested that phytohormones and UPS crosstalk might play a critical role in the wheat grain developmental process. A meta-analysis of these genes led to the identification of 26 E3 ligase candidate genes and 12 hormones-related candidate genes that are preferentially expressed in the endosperm. The functional model that we proposed and the identification of candidate genes should help to better understand wheat grain development.