Die Muscle-RING-finger (MuRF) Proteine sind E3-Ubiquitin-Ligasen, die im Muskelgewebe den Ubiquitin-Proteasom-System abhängigen Abbau von Proteinen vermitteln. MuRF1 wird in der Muskelatrophie verstärkt synthetisiert, was zu einem gesteigerten Proteinabbau und damit zum Verlust von Muskelmasse führt. Zudem sind Mäuse, denen MuRF1 fehlt vor Muskelatrophie geschützt. E3-Ubiquitin-Ligasen fungieren oftmals in Multiproteinkomplexen. Dies wurde für MuRF-Proteine bisher nicht gezeigt. Aufgrund dessen sollten neue MuRF-Multiproteinkomplex assoziierte Faktoren mittels Hefe-Zwei-Hybrid-System und SILAC AP-MS identifiziert und deren Einfluss auf die MuRF-Funktion charakterisiert werden. Es wurden sowohl neue als auch publizierte MuRF-Interaktionspartner (Iap) gefunden. Von den neu entdeckten MuRF-Iap wurde der Fokus auf WDR42A gelegt, da das Protein mit beiden Methoden identifiziert wurde und zudem funktionell hoch interessant ist. WDR42A homologe Proteine bilden zirkuläre β-Propeller Strukturen die Multiproteinkomplexe koordinieren. Die Interaktion zwischen MuRF-Proteinen und WDR42A wurde mittels Ko-IP Experimenten und Kolokalisationsstudien bestätigt. Cycloheximid-Abbau-Experimente deuten darauf hin, dass WDR42A kein MuRF1 Substrat-Protein ist. Da die MuRF-Proteine spezifisch im Muskel hergestellt werden, sollte überprüft werden ob WDR42A ebenfalls im Muskelgewebe synthetisiert wird. Es wurde gezeigt, dass WDR42A ubiquitär sowie im Muskelgewebe und in immortalisierten Muskelzellen hergestellt wird. Analog zu MuRF1 wird WDR42A in der Denervations-induzierten Skelettmuskelatrophie und der Muskelentwicklung verstärkt synthetisiert. Die Herunterregulation von WDR42A mittels siRNA in C2C12 Myotuben schützte diese Zellen vor dem Auftreten von Atrophie. Diese Ergebnisse zeigen, dass WDR42A wie MuRF1 an der Entstehung von Muskelatrophie beteiligt ist. Aufgrund der WDR42A Domänenstruktur wird vermutet, dass WDR42A als Scaffolding-Protein MuRF1-Multiproteinkomplexe reguliert. / The muscle-RING-finger (MuRF) proteins are E3 ubiquitin ligases which coordinate the ubiquitin-proteasome system dependent protein degradation in muscle tissue. MuRF1 is up-regulated under muscle atrophy conditions. This leads to enhanced proteolysis and thereby to loss of muscle mass and strength. Furthermore are MuRF1 knockout mice resistant to muscle atrophy. E3 ubiquitin ligases often operate in multi-protein complexes. This has not been shown for MuRF proteins. Therefore we used yeast-two-hybrid and SILAC-AP-MS to identify and subsequently characterize new MuRF multi-protein complex associated proteins. We found new and also published MuRF interaction partners (Iap) with both methods. Amongst the new Iap, we focused on WDR42A, because it was found with both techniques and his interesting functional potential. WDR42A exhibits seven consecutive arranged WD40-repeat domains. This domain arrangement leads in homologues proteins to the formation of seven-bladed β-propeller structures, which act as protein interaction platforms that coordinate multi-protein complexes. The protein interaction between the MuRFs and WDR42A was confirmed with Co-IP and co-localization experiments. Cycloheximide decay experiments indicated that WDR42A is not a MuRF1 substrate protein. The MuRF proteins are muscle specific, therefore we tested if WDR42A is also synthetized in muscle tissue. We could show that WDR42A is ubiquitously, but also in muscle tissue as well as in immortalized muscle cells produced. WDR42A is similar to MuRF1 up-regulated under denervation-induced skeletal muscle atrophy as well as in muscle development. Furthermore are C2C12 myotubes resistant to muscle atrophy after siRNA down-regulation of WDR42A. These results demonstrate that WDR42A is like MuRF1 important for the development of muscle atrophy. Due to the domain structure of WDR42A, we hypothesize that WDR42A regulates MuRF1 multi protein complexes as scaffolding protein.

In Saccharomyces cerevisiae nimmt die membrangebundene RING-Ub-Ligase Doa10 eine bedeutende Rolle in der Proteinqualitätskontrolle (PQC) des Endoplasmatischen Retikulums (ER) und des Nukleus ein. Doa10 katalysiert dabei die Verknüpfung K48- verbundener Ub-Ketten auf Proteine, die entweder in der ER-Membran oder löslich im Cytosol oder dem Nukleoplasma vorliegen. Diese Markierung leitet die Degradation dieser Proteine ein. Interessanterweise kooperiert Doa10, im Gegensatz zu anderen RING-Ub-Ligasen, mit zwei Ub-konjugierenden Enzymen (E2), um ihre Substrate zu prozessieren. In dieser Arbeit wird veranschaulicht, wie die beiden hochspezialisierten E2 Enzyme Ubc6 und Ubc7 sequentiell agieren, um Doa10 Substrate zu modifizieren. Zuerst wird ein einzelnes Ub-Molekül Ubc6-abhängig an ein Substrat konjugiert (Initiation). Von diesem Rest ausgehen katalysiert Ubc7 die Ausbildung einer K48-verbundenen Ub-Kette (Elongation). Die Fähigkeit von Ubc6 nicht nur Lysine, sondern auch hydroxylierten Aminosäuren wie Serin und Threonin mit Ub-Molekülen zu verknüpfen, erweitert das Substratspektrum von Doa10 und ermöglicht die Prozessieren von Proteinen, die keine zugänglichen Lysinreste exponieren. Weiterhin wird gezeigt, dass ein Überangebot von Ubc6 den Doa10-abhängigen Substratabbau beeinträchtigt. Dies weist darauf hin, dass die Generierung eines effizienten Poly-Ub-Signals einer streng kontrollierten Koordination beider E2 Enzyme am Doa10-Ligase-Komplex unterliegt. / In Saccharomyces cerevisiae, the membrane-bound RING-type Ub ligase Doa10 is a key player of Protein Quality Control (PQC) in the endoplasmic reticulum (ER) and the nucleus. Doa10 promotes lysine 48-linked poly-ubiquitylation of proteins that either reside in the ER membrane or are soluble in the cytosol or the nucleus and thereby labels them for degradation. Strikingly, in contrast to other RING Ub ligases, which typically employ a single Ub conjugating enzyme (E2) for substrate ubiquitylation, the Doa10 ligase requires two of such enzymes for client processing. This study demonstrates that the highly specialized E2 enzymes Ubc6 and Ubc7 act in a sequential manner on Doa10 client proteins. In a first step Ubc6 attaches a single Ub molecule to a substrate (priming), which is followed by the elongation of this moiety with K48-linked Ub chains by Ubc7 (elongation). The ability of Ubc6 to conjugate Ub not only to lysine but also to hydroxylated amino acids like serine and threonine broadens the substrate range of Doa10 and allows processing of proteins, which do not expose accessible lysine residues. Overproduction of Ubc6 was shown to impair Doa10 dependent substrate degradation. Apparently, the generation of a productive K48-linked poly-Ub signal requires a tightly coordinated activity of the individual E2 enzymes at the Doa10 ligase complex.

La relocalisation et la dégradation médiée par ubiquitination sont utilisées par la cellule pour contrôler la localisation et l’expression de ses protéines. L’E3 ubiquitine ligase MARCH1 est impliqué dans la régulation post-traductionnelle de CMH-II et de CD86. Dans ce mémoire, on propose un rôle additionnel à MARCH1. Nos résultats expérimentaux nous portent à croire que MARCH1 pourrait moduler le métabolisme cellulaire en favorisant la relocalisation et la dégradation d’enzymes impliquées dans la glycolyse. La grande majorité des cellules utilise la phosphorylation oxydative pour générer de l’ATP en présence d’oxygène. Dans un environnement hypoxique, cette dernière est non fonctionnelle et la cellule doit utiliser la glycolyse anaérobique pour produire son ATP. Une cellule cancéreuse à des besoins énergétiques supérieurs en raison de l’augmentation de sa biomasse et de sa prolifération incontrôlée. Pour subvenir à ces besoins, elle maximise sa production d’énergie en modifiant son métabolisme; c’est l’effet Warburg. On retrouve dans les cellules immunitaires des modifications similaires au métabolisme cellulaire suite à un signal d’activation. Ici, nous montrons que la respiration mitochondriale maximale, la réserve respiratoire et la glycolyse maximale sont diminuées dans les cellules présentatrice d’antigènes qui expriment MARCH1. Nous avons montré que MARCH1 était localisable au niveau de la mitochondrie, ce qui lui permet d’interagir avec les enzymes de la glycolyse. Finalement, nous avons quantifié l’expression de Eno1 et de LDHB par Western Blot, pour montrer une augmentation de l’expression de ces enzymes en absence de MARCH1. À la lumière de ces résultats, nous discutons des avantages que procure la diminution de l’expression de MARCH1 dans un contexte inflammatoire, suite à l’activation des cellules présentatrices d’antigènes. Ce phénomène permettrait une présentation antigénique plus efficace, une augmentation de la production d’énergie et une meilleure résistance aux ROS produits lors de la réponse inflammatoire. / Relocation and degradation mediated by ubiquitination are used by the cell to control the localization and the expression of proteins. E3 ubiquitin ligase MARCH1 is known to be involved in post-translational regulation of MHC-II and CD86. In this thesis, we suggest an additional role to MARCH1. Our experimental results lead us to believe that MARCH1 may modulate cellular metabolism by promoting the relocation and degradation of enzymes involved in glycolysis. The vast majority of cells generate ATP from oxidative phosphorylation in presence of oxygen. In a hypoxic environment, the latter is non-functional and the cell must use the anaerobic glycolysis to produce ATP. A cancerous cell requires more energy due to increased biomass and its uncontrolled proliferation. To meet these needs, it maximizes its energy production regardless of oxygen concentrations. Many studies have shown that aerobic glycolysis is preferred to oxidative phosphorylation in cancer cells, even if the two pathway are used simultaneously; it is described as the Warburg effect. Similar modification of the cellular metabolism is also found in immune cells after an activation signal to fulfill the cell functions. Here we show that the maximal mitochondrial respiration, the respiratory reserves and the maximal glycolysis are reduced in antigen-presenting cells that express MARCH1. Furthermore, we showed that MARCH1 can be localized on the mitochondria to interact with it’s target. Finally, we quantified the expression of Eno1 and LDHB by Western blot to show an increased expression of these enzymes in the absence of MARCH1. Thus, we discuss the benefits of the expression reduction of MARCH1 in an inflammatory context, following the activation of antigen presenting cells. This phenomenon would allow a better antigen presentation, an increased energy production and a greater resistance to ROS, produced during the inflammatory response.

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation.

The innate immune system is the first line of defence against pathogen invasion. The range of diseases that are caused by deficiencies in or deregulation of the innate immune system illustrates the importance of maintaining an effective balance between clearance of infectious agents and minimisation of inflammatory mediated tissue damage. This thesis explores the role of two proteins in the regulation of the innate immune system. Primarily, this work investigates the effect of human β-defensin 3 (hBD3) on the response to self-DNA and pathogenic DNA. HBD3 is an antimicrobial peptide (AMP), which has been shown to have a role in regulating the immune response; increased copy number of the region containing the gene for hBD3, DEFB103, is linked to an increased risk of psoriasis. Additionally, a similar cationic AMP, LL37, has been shown to exacerbate the pathogenesis of psoriasis by forming an immunogenic complex with self-DNA. This lead to the hypothesis that hBD3 may also affect the innate immune response to DNA. Therefore this project investigates what effect hBD3 has on the response of the innate immune system to self and pathogenic DNA. Flt-3 dendritic cells were used to show that whilst hBD3 increased cellular uptake of self-DNA, it did not convert self-DNA into an immune stimulus. However, hBD3 significantly exacerbated the response to bacterial DNA in a TLR9-dependent manner, also by increasing cellular uptake into FLDCs. The finding that hBD3 increased cellular uptake of both self- and pathogenic DNA suggests that at sites of infection or increased cell death, where DNA would be found in the extracellular environment, hBD3 may increase uptake into immune cells and could induce an increased immune response. Since increased hBD3 expression is induced by inflammatory stimuli, this process would cause a positive feedback loop of inflammation during bacterial infections. In conclusion, hBD3’s role in regulating the innate immune response to DNA is at the ligand-receptor level rather than affecting signalling pathways. Furthermore, hBD3 promotes the innate immune response to bacterial DNA by increasing the efficiency of cellular uptake possibly by inducing DNA aggregation. These results implicate a possible role for hBD3 in the earliest stages of psoriatic plaque development, which is often initiated or exacerbated by an infection, and this could be investigated further. Secondly, I investigated the innate immune function of an E3 ubiquitin ligase (E3L) not previously associated with human disease. Mutations in E3L have been identified in three microcephalic primordial dwarfism families; these patients also presented with recurrent respiratory illnesses. E3L has been implicated in the regulation of the innate immune system via interactions with signalling pathways downstream of the receptor, though its role is not clear. We hypothesised that E3L had a dual role both in regulating growth and cell division and in regulating the immune system. Primary patient fibroblasts did not demonstrate an altered cytokine response to bacterial or viral ligands, implying that E3L may have a specific function in immune cells. To investigate this further, and to provide a system to study E3L in vivo, two transgenic mouse lines were designed and engineered, firstly a conditional ‘knock-out’ designed to replicate some of the alternative isoforms of E3L seen in RT-PCRs, and secondly a ‘knock-in’ line to recapitulate the human mutation in exon 7 of E3L, R185X. These mouse lines should offer an insight into the developmental role for E3L, and contribute to establishing a potential role for E3L in the innate immune system. This thesis exemplifies the complexity of the innate immune system and the regulatory pathways that interact to maintain a delicate homeostasis preventing pathogenic inflammation. Understanding these regulatory mechanisms may shed light on the pathogenicity of diseases and identification of potential targets for therapeutics.

A via RAS/RAF/MEK/ERK é frequentemente hiperativada em diversos tumores. Em leucemias sua ativação pode ocorrer, dentre outros mecanismos, a partir de mutações pontuais nos genes da família RAS, que são relevantes nas leucemias linfóide e mielóide agudas (LLA e LMA), ou a partir da atividade da tirosina-quinase BCR-ABL, que é responsável por promover a tumorigênese na leucemia mielóide crônica (LMC) e em alguns casos de LLA. A hiperativação dessa via estimula a proliferação celular e, consequentemente, a produção de espécies reativas de oxigênio (ROS), que é um dos principais mecanismos envolvidos com a indução de senescência celular em tumores. Assim sendo, as células tumorais que apresentam o gene RAS mutado são criticamente dependentes de mecanismos de feedback para regular a ativação da via. Jang et al. demonstraram que a ubiquitina-ligase HUWE1 atua em um mecanismo de feedback negativo que controla a ativação de ERK1/2 e apesar de amplamente estudada no contexto da tumorigênese, a atuação dessa molécula em eventos relacionados à leucemogênese ainda não foi descrita. No presente estudo, linhagens celulares leucêmicas e células tronco e progenitoras hematopoiéticas humanas (HSPCs) com mutação KRASG12V foram transduzidas com partículas lentivirais miR-E para o silenciamento gênico de HUWE1. Ensaios de proliferação celular, apoptose, análise do ciclo celular, produção de ROS e análise da expressão gênica e proteica foram realizados nas linhagens celulares; análise do crescimento cumulativo, área de formação de cobblestones, capacidade clonogênica e análise do perfil de diferenciação celular foram realizados nas HSPCs. Nas linhagens celulares observouse que o silenciamento de HUWE1 reduziu a capacidade proliferativa das linhagens Nalm-6, K562 e THP-1, porém não causou nenhum prejuízo à capacidade proliferativa da linhagem HL-60. Além disso, causou a redução da produção de ROS (p<0,05), associada à redução das taxas de apoptose (p<0,01), principalmente na linhagem K562, na qual também promoveu a ativação de ERK1/2 . Em HSPCs, observou-se a redução da capacidade proliferativa das culturas que expressavam o oncogene KRASG12V associado ao silenciamento de HUWE1. Nas mesmas condições foi observada uma drástica redução na capacidade clonogênica das HSPCs (p<0,001), em especial as do tipo BFU-E. O silenciamento de HUWE1 também alterou o perfil de diferenciação celular para a linhagem monocítica. Os resultados sugerem que HUWE1 pode participar do processo de leucemogênese e diferenciação de HSPCs humanas participando na modulação da via RAS/RAF/MEK/ERK. / The RAS/RAF/MEK/ERK pathway is frequently hyperactivated in several tumors. In leukemia, this activation can arise, among other mechanisms, from point mutations in the RAS genes, which are important in acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML), or from chromosomal translocations such as the BCR-ABL gene, which is a driver mutation in chronic myeloid leukemia (CML) and some cases of ALL. The hyperactivation of this pathway stimulates cell proliferation and, consequently, the production of reactive oxygen species (ROS), which is one of the main mechanisms involved with induction of cellular senescence in tumors. Thus, tumor cells that harbor the mutated RAS gene are critically dependent on feedback mechanisms to regulate pathway activation. Jang et al. demonstrated that the ubiquitinligase HUWE1 acts on a negative feedback mechanism that controls the activation of ERK1/2. Although widely studied in the context of tumorigenesis, the role of this molecule in events related to leukemogenesis has not yet been described. In this study, leukemia cell lines and human hematopoietic stem and progenitors cells (HSPCs) with KRASG12V mutation were transduced with miR-E lentiviral particles for HUWE1 knockdown. Cell proliferation, apoptosis, cell cycle analysis, ROS production and analysis of gene and protein expression were performed in cell lines; cumulative growth analysis, cobblestones area formations, clonogenic capacity and differentiation profile analysis were performed in HSPCs. In cell lines, it was observed that HUWE1 knockdown reduced the proliferative capacity of Nalm-6, K562 and THP-1, but not of HL-60. Besides that, it caused a reduction in ROS production (p<0,05), associated with reduction of apoptosis rates (p<0,01), especially in K562 in which it also promoted activation of ERK1/2. In HSPCs, a reduction of the proliferative capacity was observed in cultures expressing KRASG12V in combination with HUWE1 knockdown. In the same conditions, a drastic reduction of clonogenic capacity (p<0,001), especially of BFU-E colonies, was observed. HUWE1 knockdown also changed differentiation profile to the monocytic lineage. Results suggest that HUWE1 might play a role in leukemogenesis process and differentiation of human HSPCs, acting in the modulation of RAS/RAF/MEK/ERK.

A via RAS/RAF/MEK/ERK é frequentemente hiperativada em diversos tumores. Em leucemias sua ativação pode ocorrer, dentre outros mecanismos, a partir de mutações pontuais nos genes da família RAS, que são relevantes nas leucemias linfóide e mielóide agudas (LLA e LMA), ou a partir da atividade da tirosina-quinase BCR-ABL, que é responsável por promover a tumorigênese na leucemia mielóide crônica (LMC) e em alguns casos de LLA. A hiperativação dessa via estimula a proliferação celular e, consequentemente, a produção de espécies reativas de oxigênio (ROS), que é um dos principais mecanismos envolvidos com a indução de senescência celular em tumores. Assim sendo, as células tumorais que apresentam o gene RAS mutado são criticamente dependentes de mecanismos de feedback para regular a ativação da via. Jang et al. demonstraram que a ubiquitina-ligase HUWE1 atua em um mecanismo de feedback negativo que controla a ativação de ERK1/2 e apesar de amplamente estudada no contexto da tumorigênese, a atuação dessa molécula em eventos relacionados à leucemogênese ainda não foi descrita. No presente estudo, linhagens celulares leucêmicas e células tronco e progenitoras hematopoiéticas humanas (HSPCs) com mutação KRASG12V foram transduzidas com partículas lentivirais miR-E para o silenciamento gênico de HUWE1. Ensaios de proliferação celular, apoptose, análise do ciclo celular, produção de ROS e análise da expressão gênica e proteica foram realizados nas linhagens celulares; análise do crescimento cumulativo, área de formação de cobblestones, capacidade clonogênica e análise do perfil de diferenciação celular foram realizados nas HSPCs. Nas linhagens celulares observouse que o silenciamento de HUWE1 reduziu a capacidade proliferativa das linhagens Nalm-6, K562 e THP-1, porém não causou nenhum prejuízo à capacidade proliferativa da linhagem HL-60. Além disso, causou a redução da produção de ROS (p<0,05), associada à redução das taxas de apoptose (p<0,01), principalmente na linhagem K562, na qual também promoveu a ativação de ERK1/2 . Em HSPCs, observou-se a redução da capacidade proliferativa das culturas que expressavam o oncogene KRASG12V associado ao silenciamento de HUWE1. Nas mesmas condições foi observada uma drástica redução na capacidade clonogênica das HSPCs (p<0,001), em especial as do tipo BFU-E. O silenciamento de HUWE1 também alterou o perfil de diferenciação celular para a linhagem monocítica. Os resultados sugerem que HUWE1 pode participar do processo de leucemogênese e diferenciação de HSPCs humanas participando na modulação da via RAS/RAF/MEK/ERK. / The RAS/RAF/MEK/ERK pathway is frequently hyperactivated in several tumors. In leukemia, this activation can arise, among other mechanisms, from point mutations in the RAS genes, which are important in acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML), or from chromosomal translocations such as the BCR-ABL gene, which is a driver mutation in chronic myeloid leukemia (CML) and some cases of ALL. The hyperactivation of this pathway stimulates cell proliferation and, consequently, the production of reactive oxygen species (ROS), which is one of the main mechanisms involved with induction of cellular senescence in tumors. Thus, tumor cells that harbor the mutated RAS gene are critically dependent on feedback mechanisms to regulate pathway activation. Jang et al. demonstrated that the ubiquitinligase HUWE1 acts on a negative feedback mechanism that controls the activation of ERK1/2. Although widely studied in the context of tumorigenesis, the role of this molecule in events related to leukemogenesis has not yet been described. In this study, leukemia cell lines and human hematopoietic stem and progenitors cells (HSPCs) with KRASG12V mutation were transduced with miR-E lentiviral particles for HUWE1 knockdown. Cell proliferation, apoptosis, cell cycle analysis, ROS production and analysis of gene and protein expression were performed in cell lines; cumulative growth analysis, cobblestones area formations, clonogenic capacity and differentiation profile analysis were performed in HSPCs. In cell lines, it was observed that HUWE1 knockdown reduced the proliferative capacity of Nalm-6, K562 and THP-1, but not of HL-60. Besides that, it caused a reduction in ROS production (p<0,05), associated with reduction of apoptosis rates (p<0,01), especially in K562 in which it also promoted activation of ERK1/2. In HSPCs, a reduction of the proliferative capacity was observed in cultures expressing KRASG12V in combination with HUWE1 knockdown. In the same conditions, a drastic reduction of clonogenic capacity (p<0,001), especially of BFU-E colonies, was observed. HUWE1 knockdown also changed differentiation profile to the monocytic lineage. Results suggest that HUWE1 might play a role in leukemogenesis process and differentiation of human HSPCs, acting in the modulation of RAS/RAF/MEK/ERK.

An intact cellular response to DNA damage is important for the maintenance of genomic stability and tumour prevention. EDD, the human orthologue of Drosophila melanogaster ???hyperplastic discs???, is over-expressed or mutated in a number of common human cancers. EDD is a progestin regulated gene that encodes an E3 ubiquitin ligase involved in cell communication and cell adhesion, and although it has also been implicated in the DNA damage response through its association with DNA damage proteins, a definitive role has yet to be demonstrated. The work presented herein shows that EDD is necessary for an adequate cellular response to double-strand DNA breaks. Cells depleted of EDD exhibit reduced survival, radio-resistant DNA synthesis and failure to maintain G2/M arrest following DNA damage induced by phleomycin exposure. Furthermore, EDD-depleted cells display impaired activating phosphorylation and kinase activity of the checkpoint kinase CHK2 after DNA damage. These effects appear to be largely modulated through a phospho-dependent interaction involving the CHK2 FHA domain and a region of EDD spanning a number of putative FHA-binding threonines. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasise the potential importance of EDD in cancer.

DNA repair pathways have evolved to protect the genome from damage caused by intrinsic and extrinsic factors. Although numerous DNA repair mechanisms have been studied and reported, information regarding how they coordinate with the necessary changes in chromatin structure is scarce. Smc (structural maintenance of chromosomes) proteins are a conserved, essential family of proteins required for chromosome organization and accurate segregation. The budding yeast, Saccharomyces cerevisiae has three Smc-protein complexes: Smc1/3 complex (cohesin), Smc2/4 complex (condensin) and the Smc5/6 complex, required for sister chromatid cohesion, condensation and DNA repair, respectively. The chromatin associated Smc5/6 complex consists of Smc5, Smc6 and six non-smc elements (Nse1-Nse6). Smc5 and Smc6 are required for stability of repetitive chromosomal regions and sister chromatid recombination-mediated repair of double-strand breaks. Mms21/Nse2, a subunit of the Smc5/6 complex, is a SUMO E3-ligase, which conjugates SUMO (small ubiquitin-like modifier) to Smc5 and Yku70 (DNA repair protein) and its SUMO ligase activity protects the cells from extrinsic DNA damage. To address the role of Nse2 SUMO ligase in cellular events, we isolated mutants (nse2∆sl and nse2C221A) defective in the E3-ligase domain of Nse2 and found that these mutants are sensitive to genotoxic agents, for example MMS, UV or bleomycin, as expected. We found that cysteine 221 present in the SP-RING domain of Nse2 is required in the function of Nse2 in resisting genotoxic stress. We found that nse2∆sl cultures are slow growing and show increased abundance of cells having 2N DNA content (indicative of a G2-M cell cycle delay or arrest) relative to wild type cells. The DNA damage checkpoint pathway is activated to a limited extent in unchallenged nse2∆sl mutant cells indicating that cells lacking the SUMO ligase activity of Nse2 incur spontaneous DNA damage. Furthermore nse2∆sl cells are exquisitely sensitive to caffeine, an agent known to override the DNA damage checkpoint in a number of organisms by inhibiting the DNA damage checkpoint transducer ATR (Homo sapiens), Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe). In order to investigate the importance of the DNA damage checkpoint pathway for nse2∆sl cells, we employed a genetic approach. We found that nse2∆sl exhibits synthetic sick interaction with mec1∆ but not tel1∆ (defective in Mec1 or Tel1 PI kinases) or mrc1∆ (defective in Mrc1 or mediator of replication checkpoint 1) indicating that the DNA damage induced Mec1 dependent checkpoint pathway is selectively required but the replication stress checkpoint pathway is dispensable for optimal growth of unchallenged nse2∆sl cells. In order to further investigate the role of Nse2 in S phase events, we used camptothecin (CPT), a drug that induces S phase specific double strand breaks. CPT inhibits topoisomerase I by trapping the covalent Top1-DNA intermediate. Collision of a DNA replication fork with such a complex results in double-strand and single-strand breaks in DNA. We found that nse2∆sl is CPT-sensitive and that nse2∆sl top1-8 has a synthetic sick phenotype. Thus, our chemical and genetic interaction studies suggest that the SUMO ligase activity of Nse2 may be required when Top1 function is compromised. Interestingly, human and yeast Top1 proteins are known to be sumoylated. Our findings suggest that MMS-induced enhancement of Top1 sumoylation in budding yeast is partially dependent on SUMO ligase activity of Nse2. Since both sumoylation and Top1 play a role in telomere maintenance, we also examined the telomere length in single as well as double mutants and found that there is slight telomere lengthening in nse2∆sl top1-8 double mutant. To gain further insight into the genetic interaction between Nse2 and other proteins which affect DNA topology, we also investigated genetic interaction of Nse2 with other topoisomerases. We found that top3-2 nse2∆sl exhibited a synthetic sick phenotype but nse2∆sl top2-4 showed partial rescue of temperature sensitivity. In order to investigate whether chromosome integrity is compromised in nse2∆sl cells we employed a YAC (yeast artificial chromosome) based assay to examine GCRs (gross chromosomal rearrangements). We found elevated levels of GCR in nse2∆sl cells compared to wild type cells. Furthermore, deletion of DNA Topoisomerase1 in nse2∆sl background selectively destabilizes a longer YAC relative to shorter YACs. We also examined the effect of varying origin number on YAC stability in nse2∆sl as well as top1∆ and nse2∆sl top1∆ cells. We found that a YAC having fewer origins is not destabilized in nse2∆sl and top1∆ single mutants but is destabilized in the nse2∆sl top1∆ double mutant. Since Nse2 is a non-SMC member of the Smc5/6 complex, we also investigated the effect of varying origin number on YAC stability in smc6-56 and smc656 top1∆ mutants. We found that the stability of a YAC is modestly compromised in the smc6-56 mutant but its derivative having fewer origins is not further destabilized, rather it seems to be stabilized. In order to gain molecular insights into the involvement of the SUMO ligase activity of Nse2 in maintenance of chromosome integrity, we examined sumoylation of specific substrates following a candidate approach. Smc5 and Yku70 are known targets of Nse2dependent sumoylation. We found that Smc6 is also sumoylated and that the MMS-induced enhancement of Smc6 sumoylation in budding yeast is partially dependent on Nse2. To understand the functional significance of Smc5 sumoylation, we mutated lysine residues of all the four predicted sumoylation sites ψKXE/D, individually as well as all four together. We found that all the single as well as quadruple mutants were weakly sensitive to MMS suggesting that these putative sumoylation sites of Smc5 may contribute towards countering MMS-induced DNA damage. Interestingly, we found that Smc5 sumoylation is enhanced when treated with MMS (methyl methane sulfonate) but not significantly with HU (hydroxyurea) and CPT (camptothecin). We also generated putative ATP-binding defective mutants in Smc5. Previous studies suggest that the ATPase motif is required for the essential function of some Smc proteins (for example, Smc1 and Smc6). We found that smc5K75E and smc5K75Q, having a mutation in the lysine residue of the conserved GXGKS motif present in the Walker A type box at the Nterminus exhibited a null phenotype implying that this conserved lysine residue is required for essential function of Smc5. In this study, employing genetic and biochemical methods, we have characterized the Nse2 SUMO ligase defective mutant and analyzed its role in the unperturbed mitotic cell cycle and in genome maintenance. We have also employed genetic methods to study the involvement of both Nse2 and DNA Topoisomerase I in maintaining genomic stability. Lastly, we have addressed the functional significance of Lysine residues of putative sumoylation sites and the conserved ATP-binding motif of Smc5 by mutational analysis. In conclusion, our study highlights an important role for the SUMO ligase activity of Nse2 in maintaining genomic stability and suggests that sumoylation of Smc5 may be important for resisting MMS-induced genotoxic stress.

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples. We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I). Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II). Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III). This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.