DYRK1A is the most studied member of the DYRK family of protein kinases, because is one of the human chromosoma 21 proteins for which changes in gene dosage result in neuropathological alterations. DYRKs are activated by autophosphorylation on a tyrosine residue in the activation loop, a one-off event that takes place during translation. Accordingly, DYRK1A would be constitutively active once is synthesized. However, DYRK1A is extremely sensitive to gene dosage, and thus it is predictable that not only its activity but also its actual protein amounts have to be tightly regulated by mechanisms not yet characterized. In the present study, the protein kinase NLK has been identified as a novel regulator of DYRK1A protein stability. DYRK1A interacts with NLK in physiological conditions. The interaction results in the phosphorylation of DYRK1A at multiple sites, which have been identified by mass spectrometry analysis. These phosphorylation events promote DYRK1A proteasome-dependent degradation. Moreover, DYRK1A degradation is induced by stimulating cells with Wnt1 or Wnt3a, or overexpressing elements of the Wnt signaling cascade such as the Frizzled-1 receptor or NLK activators such as HIPK2. In addition, DYRK1A interacts with and phosphorylates -catenin and TCF-4 and enhances -catenin-dependent transcriptional activity, at least by phosphorylation of -catenin. Thus, these results suggest that DYRK1A acts as a positive factor in the Wnt--catenin signaling pathway and NLK acts as a negative regulator by targeting both DYRK1A and TCF/LEF transcription factors for proteasome-mediated degradation. / DYRK1A es el miembro más estudiado de la familia de proteína quinasas DYRK, porque es una de las proteínas de la cromosoma humano 21 para la que cambios en la dosis génica dan lugar a alteraciones neuropatológicas. Las quinasas DYRK se activan por autofosforilación en un residuo tirosina localizado en el lazo de activación, un evento único que ocurre durante la traducción. Como consecuencia, DYRK1A sería constitutivamente activa una vez se ha sintetizado. Sin embargo, DYRK1A es extremadamente sensible a la dosis génica, y por tanto es predecible que no sólo su actividad, pero también los niveles de proteína han de estar estrictamente controlados por mecanismos reguladores que todavía no han sido caracterizados. En este trabajo, la proteína quinasa NLK ha sido identificada como un nuevo regulador de la estabilidad de DYRK1A. DYRK1A interacciona con NLK en condiciones fisiológicas, y la interacción tiene como resultado la fosforilación de DYRK1A en residuos serina/treonina, varios de los cuales han sido identificados por espectrometría de masas. La interacción con NLK y la subsecuente fosforilación promueven la degradación de DYRK1A vía el proteasoma. Además, la degradación de DYRK1A es inducida por estimulación de la células con Wnt1 o Wnt3a, o por sobreexpresión de miembros de la cascada de señalización de Wnt, como el receptor Frizzled-1 o de un activador de NLK como HIPK2. Finalmente, se ha demostrado que DYRK1A se une y fosforila -catenina y TCF-4. La fosforilación de, al menos, -catenina es responsable del incremento de la actividad transcripcional dependiente de esta proteína en presencia de DYRK1A. Todos estos resultados sugieren que DYRK1A actúa como un factor positivo en la vía de señalización Wnt--catenina y NLK actúa como un regulador negativo al inducir la degradación vía proteasoma no sólo de los factores de transcripción TCF/LEF sino también del modulador positivo DYRK1A.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 120-126.

The diverse outcomes of ubiquitination primarily relate to the flexibility of ubiquitin in forming homo- or heterotypic chains on each of its seven lysine residues which in turn stimulate distinct downstream signaling pathways. These ubiquitin signals must be selectively initiated on the substrate protein and subsequently decoded to facilitate the desired cellular function. These initiation and decoding steps often involve additional post-translational modifications and ubiquitin receptor proteins, but the enzymes and ubiquitin chains involved for many ubiquitinated substrates are not clear. Here, I have explored the initiation and decoding of ubiquitin signals, focusing on lysine-11 (K11) linked polyubiquitin chains and their role in protein degradation. I established in vitro assays to understand how K11-chains are decoded and whether these chains act as a signal for proteasome-mediated degradation. Pure homotypic K11-chains did not bind the proteasome or its associated ubiquitin binding proteins, but did bind to the mitophagy ubiquitin receptors, MyosinVI and TAX1BP1. Heterotypic K11/K48 linkages not only bound the proteasome but also stimulated degradation of the cell cycle substrate, cyclin B1. To further explore the functions of K11-chains I focused on the hypoxia inducible transcription factor (HIF) pathway, as K11-ubiquitination had been implicated in proteasome-independent degradation of the transcription factor. I established an in vitro assay to initiate HIF ubiquitination, via prolyl hydroxylation, and determine the type of ubiquitin chains involved. Recombinant HIF isoforms were rapidly hydroxylated when incubated with cell extracts. Moreover, the levels of iron and small molecule metabolites within the lysates regulated HIF hydroxylation. However, this hydroxylation was insufficient to reproducibly promote HIF ubiquitination or determine the ubiquitin chains involved. While the nature of the polyubiquitin chains formed in the HIF pathway remain elusive, my studies identify distinct roles for homotypic and heterotypic K11-polyubiquitination in proteasome-mediated degradation.

La protéine GABARAPL1 (GABARAP like 1) ou GEC1 (Glandular Epithelial Cell 1), présente de forts pourcentages d'identité avec les protéines GABARAP (GABAA Receptor-Associated Protein), GATE-16 (Golgi-Associated ATPase Enhancer of 16 kDa) et Atg8 (Autophagy-related 8) ainsi qu'une identité moindre avec les protéines de la sous-famille LC3 (Light Chain 3). GABARAPL1 est exprimée dans tous les tissus, préférentiellement dans le système nerveux central, et intervient dans le transport intracellulaire des récepteurs GABAA (Gamma-AminoButyric Acid type A receptor) et KOR (K Opioid Receptor). De plus, une faible expression du gène gabarap1 est observée dans diverses lignées cancéreuses, suggérant son implication dans la genèse et/ou la progression tumorale. La recherche de partenaires protéiques a abouti à l'identification de différentes protéines. L'interaction entre GABARAPL1 et la protéine HSP90 (Heat Shock Protein 90) nouvellement identifiée a été étudiée dans le cerveau de rat et dans les cellules MCF-7. Ainsi, nous avons montré que HSP90 protège GABARAPL1 de la dégradation par le protéasome. Par ailleurs, il a été établi qu'au cours de !'autophagie, GABARAPL1 est clivée, maturée puis conjuguée à des phospholipides. Elle co-Iocalise alors partiellement au niveau de lysosomes et d'autophagosomes. Nous avons également démontré que gabarap1 est faiblement exprimé dans les tissus tumoraux de sein et que la surexpression de la protéine FLAG-GABARAPL1-6HIS dans les cellules MCF-7 diminue considérablement leur croissance. De plus, une forte expression de gabarapl 1 est corrélée à une augmentation de la survie de patientes atteintes de cancer du sein avec envahissement ganglionnaire. / The GABARAPL1 (GABARAP like 1) protein, also named GECl (Glandular Epithelial Cell 1), displays a high percentage of identity with the GABARAP (GABAA Receptor-Associated Protein), GATE-16 (Golgi-Associated A TPase Enhancer of 16 kDa) and Atg8 (Autophagy-related 8) proteins, and a lesser identity with LC3 (Light Chain 3) family of proteins. The GABARAPL1 protein is expressed in ail tissues, predominantly in the central nervous system and is involved in intracellular transport of GABAA receptors (Gamma-AminoButyric Acid type A receptor) and KOR (K Opioid Receptor). In addition, a Iow expression of the gabarap1 gene was shown in various cancer cell lines, suggesting its involvement in the genesis and/or progression oftumors. The search for GABARAPL 1 prote in partners has Ied to the identification of different proteins. The interaction between GABARAPL1 and the protein partner: HSP90 (Heat Shock Protein 90) was studied in rat brain and MCF-7 cells, in which we showed that HSP90 protects GABARAPL1 from degradation by the proteasome. Additionally, it was established that during autophagy, GABARAPL1 is cleaved to its mature form and conjugated to phospholipids. lt then co-localizes partially with lysosomes and autophagosomes. We also demonstrated that gabarap!J is weakly expressed in breast tumor tissues and that overexpression of the FLAG­GABARAPL1-6HIS recombinant protein in MCF-7 cells significantly reduces their growth. Finally, a strong expression of gabarap1 is correlated with increased survival of patients with lymph node-positive breast cancer.

Philosophiae Doctor - PhD / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice,RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners,this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen.In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors Y-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70).The results of this work suggest that, at least in the case of YB-1 and zBTB38,RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6’s involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.

Philosophiae Doctor - PhD / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice, RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners, this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen. In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors V-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70). The results of this work suggest that, at least in the case of YB-1 and zBTB38, RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6's involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.

Le syndrome de l’intestin irritable (SII) est un trouble fonctionnel d’origine multifactorielle, impliquant des facteurs environnementaux tels que le stress, l’alimentation et met en jeu un dysfonctionnement de l’axe intestin-cerveau, une micro-inflammation, une dysbiose et une hyperperméabilité intestinale. Le rôle du protéasome dans la régulation de la barrière intestinale au cours du SII a été étudié. De plus, ces troubles fonctionnels intestinaux (TFI) ont également été décrits comme exacerbés chez des patients souffrant d’obésité, dont la physiopathologie est complexe. Néanmoins, les mécanismes impliqués dans cette association restent mal compris et ont donc été recherchés. Dans ce travail, des modèles murins « SII-like » comme le modèle de stress « water avoidance stress » ou WAS et le modèle post-inflammatoire « post-TNBS » ont été utilisés afin d’étudier l’impact d’une inhibition du protéasome sur la régulation de la perméabilité intestinale. L’inhibition pharmacologique du protéasome par le PR-957 ou l’utilisation de souris invalidées pour une sous unité β2i du protéasome limite l’hyperperméabilité intestinale. Une supplémentation orale en glutamine permet également de diminuer la perméabilité intestinale. Une étude protéomique au niveau colique des souris WAS et une étude de l’ubiquitome colique de patients souffrant de SII à profil diarrhéique confirment l’implication du protéasome dans la physiopathologie du SII. Nous avons ensuite cherché à comprendre le lien entre l’obésité et le SII en combinant des modèles d’obésité (génétique et induite par une alimentation riche en graisses ou HFD) et le modèle WAS. Seules les souris HFD présentent une exacerbation de l’hyperperméabilité intestinale et une corticostéronémie plasmatique élevée en réponse au modèle WAS. Des études complémentaires suggèrent que ces résultats sont indépendants de la leptine, de la glycémie et du microbiote intestinal. Nos travaux proposent donc de nouvelles pistes de prise en charge des patients souffrant de SII, par intervention nutritionnelle via la glutamine ou en utilisant le protéasome comme cible thérapeutique. Nous suggérons également un rôle de l’alimentation (riche en graisse) dans le développement des TFI au cours de l’obésité. / Irritable bowel syndrome (IBS) is a multifactorial functional disorder, involving environmental factors (stress and diet for instance), gut-brain-axis dysfunction, micro-inflammation, dysbiosis and an alteration of intestinal permeability. The role of the proteasome in the regulation of the intestinal barrier during IBS has been studied. In addition, these intestinal functional disorders have also been described in patients with obesity. Nevertheless, the mechanisms underlying an association of intestinal functional disorders in the obesity context, remain poorly understood and have therefore been investigated in this thesis. In this study, "IBS-like" mouse models such as water avoidance stress (WAS) and the post-inflammatory (post-TNBS) models, were used to study the impact of proteasome inhibition on the regulation of intestinal permeability. We found that the pharmacological inhibition of the proteasome (with PR-957) or the use of knock-out mice for a subunit of the proteasome (β2i -/-) limit intestinal hyperpermeability occured in IBS-Like models. Moreover, we found that oral supplementation with glutamine also reduces intestinal hyperpermeability, wich, thus, can be considered as a putative nutritional treatment for IBS. A colonic proteomic study of WAS mice and a study of colonic ubiquitoma in IBS patients with diarrheal profiles confirmed the involvement of proteasome in the pathophysiology of IBS. Therefore, the link between obesity and IBS was examined by combining models of obesity (ob/ob genetic and high-fat diet [HFD] models) with WAS model. Only HFD mice displayed enhanced intestinal hyperpermeability and higher plasma corticosterone levels in response to WAS. Further studies suggest that these results, themselve, are independent of leptin, glycaemia and gut microbiota. This study paves new ways of treating patients suffering from IBS, by nutritional intervention via glutamine or by using the proteasome as a therapeutic target. We also suggest a role of diet (high fat) in the development of intestinal functional disorders during obesity.

and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...

Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...