Tesis sobre el tema "RNF216"

Gene dosage alterations of the kinase DYRK1A are linked to disease in humans. To better understand DYRK1A activities an interactome analysis was performed. RNF169, an E3-ubiquitin ligase key component of the cellular response to double-strand breaks (DSBs), was found as a top nuclear interactor. The functional characterization of this interaction has uncovered that a dedicated motif in the non-catalytic N-terminus of DYRK1A is responsible of the direct interaction with RNF169 and that this interaction is essential for the recruitment of DYRK1A to DSBs. Using a combination of mass spectrometry analysis, mutagenesis, and in vitro kinase assays several DYRK1A-dependent phosphosites have been identified in RNF169 and its paralog RNF168. Reporter-cell assays and IRIF analysis showed that DYRK1A silencing perturbs the DSB-repair pathways. In agreement, DYRK1A knockdown leads to increased radiation sensitivity. All together, the data suggest a role for DYRK1A in DSB-repair that might involve the phosphorylation of RNF168 and RNF169.
Alteraciones de la dosis génica de la quinasa DYRK1A son causantes de enfermedad en humanos. Para profundizar en las actividades biológicas de DYRK1A, se ha realizado un estudio de interactoma, en el que RNF169, elemento clave en la respuesta al daño al DNA causado por roturas de doble cadena, se reveló como uno de los principales interactores. La interacción DYRK1A-RNF169 es directa y responsable de la localización de DYRK1A en el DNA en respuesta al daño. La combinación de espectrometría de masas, mutagénesis y ensayos quinasa ha permitido identificar varios residuos fosforilados por DYRK1A en RNF169 y en su parálogo RNF168, que actúa en el mismo proceso. El silenciamiento de DYRK1A causa alteraciones en los mecanismos de respuesta al daño y las células presentan un aumento de la sensibilidad a la radiación. Estos resultados permiten sugerir que DYRK1A puede ser un nuevo regulador de la respuesta al daño al DNA.

Background: Breast cancer is the commonest female cancer. DNA double-strand breaks (DSBs) associated proteins such as BRCA1 have been shown to be involved in tumourigenesis of breast tissue. One of the key regulators of DSBs, the RING Finger Protein 168 (RNF168), controls DNA damage responses (including the manipulation of homologous recombinant and non-homologous end-joining repair) which are responsible for correction of errors that occur during DSBs in order to maintain genomic stability. The nature of this protein suggests that RNF168 may play an important role in development of breast cancer. Material and methods: This study investigated the relationship of RNF168 expression in breast cancer by immunohistochemistry staining of 118 breast cancer samples in tissue microarray. The nuclear stain and cytoplasmic stain of the sections were assessed. Nuclear localization score was obtained and correlated with clinico-pathological features of the patients. Results: Immunohistological staining of RNF168 was successful in 99 cases of the tested breast cancer specimens. The expression of RNF168 was found to be significantly correlated with the occurrence of breast cancer metastasis (p=0.032). Strong expression of the protein was also found to be significantly associated with poorer breast cancer prognosis (p=0.033). In addition, correlation analysis also showed marginal correlation between nuclear localization of RNF168 with the age of patients at their first disease diagnosis (p=0.061). Conclusion: RNF168 might play a critical role in promoting breast cancer metastasis during the advanced stage of breast cancer, which results in poor disease prognosis. Detailed mechanism involved in metastasis promotion remained to be revealed in further study.
published_or_final_version
Pathology
Master
Master of Medical Sciences

Le gène RNF213 a été identifié comme un facteur de risque associé au développement de maladies cérébrovasculaires (MCV) notamment, la maladie de Moyamoya (MMM) et les anévrismes intracrâniens (AIC). Malgré une incompréhension des fonctions biologiques exactes, la ring finger protein 213 (RNF213) serait impliquée dans la régulation de la prolifération cellulaire, de l’angiogenèse et de l’inflammation. Les travaux présentés dans cette thèse se concentrent sur le développement de modèles vasculaires in vitro afin de mieux caractériser le rôle de RNF213 dans MCV. L’hypothèse est que l’invalidation de la protéine RNF213 dans des cellules endothéliales (CE) cerébrales pourrait recréer certains phénotypes associés au développement de la MMM et à la formation d’AIC. Des cellules endothéliales microvasculaires humaines de cerveau (hCMEC/D3) invalide en RNF213 (RNF213-/- ) ont été initialement générées par la méthode Clustered Regularly Interspaced Short Palindromic Repeats et la protéine associée Cas9 (CRISPR-Cas9) double nickase. La première partie des travaux porte sur le rôle que jouerait RNF213 dans l’homéostasie de la barrière hémato-encéphalique (BHE) et dans les étapes précoces de la pathogenèse associée à la MMM. Plus précisément, la perte des jonctions adhérentes provoquée par l’invalidation de RNF213 dans les hCMEC/D3 a été évaluée in vitro sur plusieurs paramètres, tels que la morphologie endothéliale, l’expression génique des protéines de jonctions, la localisation cellulaire, la perméabilité, l’infiltration immunitaire et le sécrétome des cytokines inflammatoires. Les résultats ont démontré que la déficience en RNF213 provoque une diminution de l’expression de la platelet endothelial cell adhesion molecule-1 (PECAM-1) qui affecte conséquemment la formation adéquate du complexe jonctionnel. De plus, une diminution de l’expression des gènes de la claudine-5, de la b-caténine et de la plakoglobine a été mesurée. La perte de RNF213 est également accompagnée d’un relargage de plusieurs cytokines proinflammatoires. En deuxième lieu, les travaux de cette thèse ont également démontré que RNF213 joue un rôle prépondérant dans le processus angiogénique des hCMEC/D3. Ceci a été étudié sous plusieurs angles d’approches, tels que la prolifération et la migration cellulaire, la formation de micro-capillaires sur un support Matrigel® et dans un modèle tridimensionnel (3D) reconstruit par génie tissulaire, l’expression génique et le sécrétome angiogénique. Les résultats ont démontré une diminution du taux de division cellulaire et une augmentation de la migration. Les études in vitro ont démontré également, pour la première fois, une augmentation significative de la formation de micro-capillaires et de la sécrétion abondante de facteurs pro-angiogénique, tels que le vascular endothelial growth factor (VEGF). Plus précisément, les hCMEC/D3 déficientes en RNF213 forment un réseau plus vaste, dense et étendu de micro-capillaires sur un support de Matrigel®. iii Lorsqu’ensemencées dans un modèle 3D plus complexe structurellement, les hCMEC/D3 forment un réseau pouvant s’apparenter au réseau de capillaires compensatoire retrouvé chez les patients MMM. Dans l’ensemble, l’invalidation du gène RNF213 dans un modèle in vitro 3D de cellules endothéliales cérébrales permet de reproduire certains phénotypes pathologiques de la MMM et devient donc ainsi le 1er modèle in vitro pour l’étude de cette maladie et des autres maladies associées à RNF213. Finalement, nous avons mis au point un nouveau modèle de vaisseaux sanguins de petit calibre reconstruit par génie tissulaire (TEBV) pour son utilisation dans l’étude in vitro de maladies vasculaires et de MCV complexes. L’ensemencement de fibroblastes ou de cellules musculaires lisses (CML) directement sur un mandrin de polyéthylène téréphtalate glycol (PETG) prétraité aux rayons ultraviolets C (UV-C) a permis de former des feuillets circulaires, manipulables et superposables. Avec cette technique, nous avons généré des TEBV complets avec les trois principales couches, soit l’adventitia, la media et l’intima tunica, qui possèdent des propriétés histologiques et mécaniques similaires aux artères humaines natives. Ce modèle optimisé et uniformisé de TEBV permettra de modéliser des pathologies vasculaires complexes, telles que la MMM et les AIC. En effet, la génération de vaisseaux complets à partir de cellules pathologiques ou de cellules éditées génétiquement pourrait faciliter la caractérisation de la pathogenèse et aider au développement de médicaments.
RNF213 has been associated as a susceptibility gene for the development cerebrovascular diseases (CVDs), in particular, moyamoya disease (MMD) and intracranial aneurysms (ICA). While the exact biological functions of RNF213 remain to be demonstrated, it is known to be involved in the regulation of cell proliferation, angiogenesis and inflammation. The work presented in this thesis focuses on the development of vascular models in vitro to better characterize the role of RNF213 in CVDs. The hypothesis is that the complete invalidation of the RNF213 protein in brain endothelial cells (EC) could recreate evident phenotypes associated with the development of MMD and the formation of ICA. We have initially generated human brain microvascular endothelial cells (hCMEC/D3) deficient in RNF213 (RNF213-/- ) using the robust CRISPR-Cas9 double nickase method. At first, our work described the role that RNF213 would play in the homeostasis of the blood-brain barrier (BBB) maintenance and in the early stages of MMD pathogenesis. More specifically, the loss of adherent junctions caused by the invalidation of RNF213 in hCMEC/D3 was evaluated in vitro on several parameters, such as endothelial morphology, gene expression of junctional proteins, cellular localization, permeability, immune infiltration and the secretome of inflammatory cytokines. Our data demonstrated that RNF213 deficiency provokes a significant decrease in the platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which consequently affects the proper formation of the junctional complex. A decrease in the expression of the claudin-5, b-catenin and plakoglobin genes was also measured. In addition, RNF213 loss is accompanied with a release of several pro-inflammatory cytokines. Thereafter, the present work also demonstrated that RNF213 plays a preponderant role in the angiogenic process of hCMEC/D3. Angiogenesis has also been characterized on several aspects, such as proliferation, migration, formation of micro-capillaries on a Matrigel®-based support and in a 3D model reconstructed by tissue engineering, gene expression and secretion of angiogenic factors. Our data demonstrates a decrease in cell division rate and an increase in cell migration. In vitro studies have also shown, for the first time, a significant increase in micro-capillary formation and abundant secretion of pro-angiogenic factors, such as the vascular endothelial growth factor (VEGF). More precisely, the hCMEC/D3 deficient in RNF213 forms a wider, denser and more extensive network of micro-capillaries on a Matrigel®-based support. When seeded in a more structurally complex 3D model, hCMEC/D3 form a network that can resemble to the compensatory capillary network found in MMM patients. Overall, the invalidation of the RNF213 gene in a 3D in vitro model of cerebral endothelial cells makes it possible to reproduce certain pathological phenotypes of MMM and v therefore becomes the 1st in vitro model for the study of this disease and other diseases associated with RNF213. Finally, we developed a new model of small-caliber blood vessels reconstructed by tissue engineering (TEBV) to be used to study vascular diseases and complex CVD in vitro. The direct seeding of fibroblasts or smooth muscle cells (CML) onto a polyethylene terephthalate glycol (PETG) mandrel that was pretreated with ultraviolet C (UV-C) radiation facilitate the formation of circular cell sheets, which could be manipulated and stacked in top of each other. Using this novel technique, we were able to successfully generate complete TEBVs with the three main arterial layers: the adventitia, the media and the intima tunica. Taken together, our TEBV model has histological and mechanical properties similar to native human arteries. Furthermore, this optimized and standardized 3D vascular construct will accelerate the scientific progress to modelized complex vascular pathologies, such as MMD and AIC. Indeed, the generation of complete vessels derived from pathological cells or genetically edited cells could facilitate the characterization of pathogenesis and help in the development of drugs.

Eukaryotic cells rely on quality control mechanisms to sustain protein homeostasis by regulating protein folding, targeting and degradation. These mechanisms involve the recognition of exposed hydrophobic regions of membrane proteins that have mislocalized to the cytosol to prevent them from misfolding and aggregation. In mammals, the heterotrimeric BAG6 complex, composed of three proteins, BAG6, UBL4A and TRC35, work closely with the co-chaperone SGTA to triage hydrophobic protein clients ensuring their delivery to the ER or to the proteasomal degradation pathway. Recently, RNF126 has been identified as the BAG6-dependent E3 ligase, which ubiquitinates lysine residues on BAG6 associated substrates. However, the decision-making process in classifying hydrophobic protein clients for degradation or rescue are unclear. Therefore, the characterisation of proteins involved in triage system is critical to understanding the mechanisms of protein sorting. The work presented here provides insights into functions of the co-chaperone SGTA and the E3 ubiquitin ligase, RNF126, within the BAG6 quality control module. It also focuses on the additional role of the co-chaperone SGTA and its yeast homologue Sgt2, which is the interaction with molecular chaperones such as Hsp70 (Ssa1 in yeast) and Hsp90 (Hsc82 in yeast) through the central tetratricopeptide repeat (TPR) domain. The solution structures of the N-terminal dimerisation domain of SGTA and N-terminal zinc finger motif of RNF126 E3 ligase are presented in Chapter 3 and 4 respectively. The interactions of both RNF126 and SGTA with the UBL domains of BAG6 and UBL4A are also characterised. This includes the structural models of complexes of RNF126_NZF and SGTA_NT with UBLs using NMR spectroscopy and HADDOCK (Chapters 3 and 4). Chapter 5 contains the x-ray structures of Sgt2_TPR and its complex with the extreme C-terminal of Ssa1. In addition, it shows that Sgt2_TPR interacts with C-terminal fragments of Ssa1, Hsc82 and Ybr137wp (a protein whose function is yet to be elucidated) in the similar binding mode and with comparable binding affinities. The binding studies were performed using biophysical methods, such as isothermal titration calorimetry and microscale thermophoresis. Together, this work aims to extend our understanding of the quality control mechanism in yeast and mammals, by providing molecular details of the components of the SGTA/BAG6 complex quality control module.

- Introducció: La meiosi és un tipus de divisió cel·lular íntimament lligat a la gametogènesi en eucariotes superiors, la finalitat és la reducció del nombre cromosòmic de diploide a haploide (és a dir, de 2n a n) en el nucli dels gàmetes. En la present tesi s'han analitzat els processos de sinapsi i recombinació en tres espècies de mamífers: humans, moixos i cans. - Contingut de la investigació: Per a l’anàlisi dels processos de sinapsi i recombinació en tres espècies de mamífers (humans, moixos i cans) s'ha utilitzat la tècnica d’immunocitogenètica, la qual ha permès determinar els valors quantitatius i les característiques qualitatives d'aquests processos. - Conclusió: La infertilitat atribuïble a alteracions en processos de recombinació i sinapsi cromosòmica en autosomes i cos sexual es restringeix a aquells individus que presenten desviacions extremes respecte del rang control d'aquests paràmetres. Existeix un fenotip meiòtic recurrent, caracteritzat per bloqueig en la transició zigotépaquité. L'aplicació de tècniques de NGS (anàlisi de l'exoma) en aquest individu infèrtil ha permès localitzar una mutació en el gen TEX11, probablement patogènica i responsable de les característiques associades a aquest fenotip. L'anàlisi de la relació de les variants al·lèliques del gen PRDM9 amb problemes de fertilitat i generació de síndromes de novo no va detectar una associació significativa en cap cas. La influència del gen RNF212 sobre la variabilitat interindividual de la recombinació observada en el present estudi està associada a la presència de polimorfismes genètics en aquest gen. Així, cada còpia de l'al·lel T en l'SNP rs3796619 d'aquest gen suposa una disminució mitjana de la taxa de recombinació de 132,5 cM en comparació amb l'al·lel C. Hi ha una relació complexa entre els genotips de PRDM9 i la taxa de recombinació, que sembla estar determinada per la longitud dels al·lels, per l'estat de homozigositat o heterozigositat dels mateixos i per efectes de dominància. Hi ha una gran variabilitat intraindividual dels aspectes quantitatius i qualitatius de la recombinació i sinapsi en les tres espècies analitzades. Aquesta variabilitat suggereix que, en mamífers, aquests processos i els factors que els controlen han de ser prou flexibles per permetre generar diversitat, encara que dins d'uns marges que garanteixin l'estabilitat genòmica. S'han observat diferències notables en els processos de recombinació entre humans i gats. Així, en gats s'ha determinat l'absència de pics de recombinació en les regions subtelomèriques, un menor efecte inhibidor del centròmer i una distribució més uniforme al llarg dels braços cromosòmics. Aquestes dues últimes observacions estan relacionades amb la menor interferència observada en aquesta espècie. S'han observat característiques sinàptiques pròpies de gats mascle, no descrites fins al moment, com la presència d'un reservori de proteïna SYCP3 des de leptoté fins paquité inicial, la separació prematura dels elements laterals a partir de paquitè tardà o la morfologia pròpia i canviant del cos sexual que, igual que en humans, pot associar-se al subestadi d'aquesta fase meiòtica. S'han detectat anomalies sinàptiques en humans, gats i gossos, encara que la incidència de asinapsis, gaps i MSUC va variar entre espècies. A més, s'ha descrit per primera vegada en aquestes tres espècies el fenomen de MSUC en individus sense alteracions cromosòmiques de poblacions salvatges. En el cos sexual, s'ha detectat presència de proteïna SYCP1 més enllà de la regió PAR a les tres espècies analitzades. Aquesta presència podria relacionar-se amb un procés de polimerització per defecte de la proteïna SYCP1, que ajudaria a la reparació dels DSBs situats al cromosoma X.
- Introducción: La meiosis es un tipo de división celular íntimamente ligado a la gametogénesis en eucariotas superiores, la finalidad es la reducción del número cromosómico de diploide a haploide (es decir, de 2n a n) en el núcleo de los gametos. En la presente tesis se han analizado los procesos de sinapsis y recombinación en tres especies de mamíferos: humanos, gatos y perros. - Contenido de la investigación: Para el análisis de los procesos de sinapsis y recombinación en tres especies de mamíferos (humanos, gatos y perros) se ha utilizado la técnica de inmunocitogenética, la cual ha permitido determinar los valores cuantitativos y las características cualitativas de estos procesos. - Conclusión: La infertilidad atribuible a alteraciones en procesos de recombinación y sinapsis cromosómica en autosomas y cuerpo sexual se restringe a aquellos individuos que presentan desviaciones extremas respecto del rango control de estos parámetros. Existe un fenotipo meiótico recurrente, caracterizado por bloqueo en la transición zigoteno-paquiteno. La aplicación de técnicas de NGS (análisis del exoma) en este individuo infértil ha permitido localizar una mutación en el gen TEX11, probablemente patogénica y responsable de las características asociadas a este fenotipo. El análisis de la relación de las variantes alélicas del gen PRDM9 con problemas de fertilidad y generación de síndromes de novo no detectó una asociación significativa en ningún caso. La influencia del gen RNF212 sobre la variabilidad interindividual de la recombinación observada en el presente estudio está asociada a la presencia de polimorfismos genéticos en ese gen. Así, cada copia del alelo T en el SNP rs3796619 de este gen supone una disminución media de la tasa de recombinación de 132,5 cM en comparación con el alelo C. Existe una relación compleja entre los genotipos de PRDM9 y la tasa de recombinación, que parece estar determinada por la longitud de los alelos, por el estado de homozigosidad o heterozigosidad de los mismos y por efectos de dominancia. Hay una gran variabilidad intraindividual de los aspectos cuantitativos y cualitativos de la recombinación y sinapsis en las tres especies analizadas. Esta variabilidad sugiere que, en mamíferos, estos procesos y los factores que los controlan deben ser lo suficientemente flexibles para permitir generar diversidad, aunque dentro de unos márgenes que garanticen la estabilidad genómica. Se han observado diferencias notables en los procesos de recombinación entre humanos y gatos. Así, en gatos se ha determinado la ausencia de picos de recombinación en las regiones subteloméricas, un menor efecto inhibidor del centrómero y una distribución más uniforme a lo largo de los brazos cromosómicos. Estas dos últimas observaciones están relacionadas con la menor interferencia observada en esta especie. Se han observado características sinápticas propias de gatos macho, no descritas hasta el momento, como la presencia de un reservorio de proteína SYCP3 desde leptoteno hasta paquiteno inicial, la separación prematura de los elementos laterales a partir del paquiteno tardío o la morfología propia y cambiante del cuerpo sexual que, al igual que en humanos, puede asociarse al subestadio de esta fase meiótica. Se han detectado anomalías sinápticas en humanos, gatos y perros, aunque la incidencia de asinapsis, gaps y MSUC varió entre especies. Además, se ha descrito por primera vez en estas tres especies el fenómeno de MSUC en individuos sin alteraciones cromosómicas de poblaciones salvajes. En el cuerpo sexual, se ha detectado presencia de proteína SYCP1 más allá de la región PAR en las tres especies analizadas. Esta presencia podría relacionarse con un proceso de polimerización por defecto de la proteína SYCP1, que ayudaría a la reparación de los DSBs situados en el cromosoma X.
- Introduction: Meiosis is a type of cell division intimately linked to gametogenesis in higher eukaryotes, the aim is the reduction of the chromosome number from diploid to haploid (from 2n to n) in the nucleus of the gametes. In the present thesis, we have analysed the processes of synapsis and recombination in three species of mammals: humans, cats and dogs. - Content of the research: For the analysis of synapsis and recombination in three species of mammals (humans, cats and dogs) the immunocytogenetic technique has been used. It allowed to determine the quantitative values and the qualitative characteristics of these processes. - Conclusion: Infertility attributable to alterations in processes of recombination and chromosomal synapsis in autosomes and the sexual body is restricted to those individuals who present extreme deviations from the control range of these parameters. There is a recurrent meiotic phenotype characterized by an arrest in the zygotene to pachytene transition. The application of NGS techniques (exome analysis) in this infertile individual has allowed to locate a mutation in the TEX11 gene, probably pathogenic and responsible for the characteristics associated with this phenotype. The analysis of the relationship of the allelic variants of the PRDM9 gene with fertility problems and generation of de novo syndromes did not detect a significant association in any case. The influence of the RNF212 gene on the interindividual variability of recombination observed in the present study is associated with the presence of a genetic polymorphisms in that gene. Thus, each copy of the T allele in the SNP rs3796619 of this gene implies a mean decrease in the recombination rate of 132.5 cM compared to the C allele. There is a complex relationship between the PRDM9 genotypes and the rate of recombination, which appears to be determined by the length of the alleles, by the homozygosity or heterozygosity, and by dominance effects. There is significant intraindividual variability of the quantitative and qualitative aspects of recombination and synapsis in the three species analysed. This variability suggests that, in mammals, these processes and the factors controlling them must be flexible enough to generate diversity, albeit within margins that guarantee genomic stability. Significant differences have been observed in recombination processes between humans and cats. Thus, in cats the absence of recombination peaks has been determined in the subtelomeric regions, a lower inhibitory effect of the centromere and a more uniform distribution along the chromosome arms. These last two observations are related to the lower interference observed in this species. Synaptic characteristics of male cats, not previously described, have been observed. These include the presence of a reservoir of SYCP3 protein from leptotene to initial pachytene, premature separation of lateral elements from late pachytene or the changing morphology of the sexual body, which, as in humans, may be associated with the substage of this meiotic phase. Synaptic abnormalities have been detected in humans, cats and dogs, although the incidence of asynapsis, gaps and MSUC varied between species. In addition, the MSUC phenomenon has been described for the first time in these three species in individuals without chromosomal alterations of wild populations. In the sexual body, SYCP1 protein has been detected beyond the PAR region in the three species analysed. This presence could be related to a default polymerization process of SYCP1 protein, which would aid in the repair of DSBs located on the X chromosome.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

Myelodysplastic syndromes (MDS) include a spectrum of stem cell malignancies characterized by ineffective hematopoiesis and predisposition to acute myeloid leukemia (AML) transformation. Patients are predominantly older (greater than 60 years old), with progressive cytopenias resulting from ineffective and cytologically dysplastic hematopoiesis. MDS subtypes are classified by morphologic features and bone marrow blast percentage, as well as cytogenetic pattern, as is the case for deletion 5q MDS. Interstitial deletion of the long arm of chromosome 5, del(5q), is the most common chromosomal abnormality in patients with MDS, and the 5q- syndrome, represents a distinct subset of del(5q) MDS characterized by an isolated deletion, megakaryocyte dysplasia, hypoplastic anemia, and an indolent natural history. MDS risk stratification is most commonly based on the International Prognostic Scoring System (IPSS) with survival outcomes ranging from a few months to many years based on risk factors. There are several therapeutic options for MDS including hematopoietic growth factors, immunosuppressive therapy, azanucleosides, and allogeneic stem cell transplant, however, there is still a need for more effective treatment options, particularly targeted therapeutics. One of the most effective treatments for MDS is selective for del(5q) MDS, and is the second generation immunomodulatory agent, lenalidomide (LEN). LEN is an analog of the known teratogen, thalidomide, and has broad biological effects including selective cytotoxicity to del(5q) clones, activation of T-cells, and expansion of erythroid precursors. In patients with del(5q) MDS, LEN is effective in up to 75% of patients, however, 50% of patients will become resistant within 2-3 years of treatment response. Studies in normal hematopoietic progenitors have shown that LEN induces expansion of the primitive erythroid precursors, which our laboratory has shown is accompanied by sensitization of progenitors to ligand induced erythropoietin receptor (EpoR) signaling. This sensitization is evidenced by increased and prolonged activation of the Signal Transducer and Activator of Transcription 5 (STAT5), compared to Epo stimulation alone. Although EpoR signaling is augmented by LEN, the exact mechanisms by which this is mediated to result in erythroid expansion are not fully characterized. In del(5q) MDS, we have shown that LEN selectively suppresses del(5q) clones via inhibition of the haploinsufficient phosphatases Cdc25c and PP2a, as well as stabilizing the human homolog of the murine double minute-2 protein (MDM2) to decrease expression of the tumor suppressor, p53, however, the mechanisms of action of LEN in non-del(5q) MDS remains elusive. Although most anemic MDS patients have normal or elevated endogenous levels of Epo, as well as comparable levels of progenitor EpoR density relative to healthy individuals, the biologic pathology underlying the impaired EpoR signaling in MDS is poorly defined. Recent reports have shown that membrane microdomains are important for T-cell, c-kit, and integrin signaling, however, there have been no reports on EpoR membrane localization. Lipid rafts are discrete membrane entities that provide platforms by which receptors aggregate and initiate downstream signaling. Furthermore, reports have indicated that there is a decrease in lipid raft density in GM-CSF primed MDS neutrophils, that consequently impaired production of reactive oxygen species (ROS) after fMLP stimulation, suggesting a role of rafts in MDS disease biology. Based on the role of rafts in signaling, and potential role in MDS pathogenesis, we sought to determine whether there was specific membrane localization of EpoR to the raft fractions, and whether disruption of rafts in MDS erythroids could impair EpoR signaling. To address this, we first examined the membrane localization of EpoR on the cell surface. We show here that EpoR translocates to lipid rafts in both erythroid progenitor cell lines as well as primary progenitor cells after stimulation by Epo. Furthermore, we found that Epo stimulation increases the assembly of lipid rafts, as well as the aggregation of rafts on the cell surface. Epo stimulation not only promoted the recruitment of EpoR into the raft fractions, but also downstream signaling intermediates such as Janus kinase 2 (Jak2), STAT5, and Lyn kinase. Moreover, a negative regulator of EpoR signaling, the CD45 tyrosine phosphatase, was redistributed outside of raft fractions after Epo stimulation, potentially enhancing receptor signal competence. Furthermore, disruption of lipid rafts by depletion of membrane cholesterol with MâCD (methyl-β-cyclodextrin) inhibited EpoR signaling in both cell lines and primary bone marrow progenitor cells. Additionally, we found that inhibition of Rho-associated, coiled-coil containing protein kinase (ROCK) and/or Ras-related C3 botulinium toxin substrate 1 (Rac1), blocked the recruitment of the receptor into the raft fractions indicating a critical role of these GTPases, and associated proteins, in the transport and localization of EpoR into raft microdomains. We next asked whether LEN could alter lipid raft assembly in erythroid precursors in the absence of Epo. LEN not only induced raft formation and aggregation but also increased F-actin polymerization. Similar to Epo stimulation, LEN alone was able to induce the recruitment of EpoR, Jak2, and STAT5 into raft fractions. Additionally, CD45 was redistributed outside of raft fractions after LEN treatment. Similarly, inhibition of ROCK blocked LEN induced raft formation and F-actin polymerization, indicating that LEN utilized effectors shared by Epo. Furthermore, LEN was able to increase raft density in raft deficient primary MDS erythroid progenitors. These data demonstrate that LEN may enhance erythroid expansion via induction of EpoR signaling competent raft platforms, to enhance survival and differentiation transcriptional response. Recently, ribosomal protein (RP), S-14, gene (RPS14) haplodeficiency was found to be a key determinant of the hypoplastic anemia in del(5q) MDS. Allelic loss of RPS14 compromises ribosome assembly, thereby causing nucleolar stress and release of free RPs that bind to and promote the degradation of MDM2, the principal negative regulator of p53. As a result, the accumulation of RPs causes lineage restricted stabilization of p53 in erythroid precursors. Our laboratory and colleagues confirmed that cellular p53 expression levels were elevated in del(5q) erythroid precursors, and that LEN decreased expression in responding patients. However, at the time of LEN treatment failure, p53 expression was again elevated at levels exceeding those at baseline. These results suggest that LEN is initially able to reverse p53 accumulation levels and that this action may be a mechanism by which LEN is selectively cytotoxic to del(5q) clones. Subsequent studies showed that LEN inhibits the cereblon E3 ubiquitin ligase complex, the newly discovered target of LEN. Cereblon has been reported to be the principal protein involved in thalidomide induced teratogenicity. Furthermore, the cytotoxic activity of LEN in multiple myeloma is dependent on cereblon. Our laboratory found that LEN inhibits the auto-ubiquitination of MDM2, thereby stabilizing the protein, and promoting ubiquitination of and ultimately the degradation of p53. Additionally, we found that LEN blocked the binding of free ribosomal proteins to MDM2, which are liberated from the nucleosome by ribosomal stress from RPS14 haploinsufficiency, consequently stabilizing the E3-ubiquitin ligase and fostering p53 degradation. In non-del(5q) MDS there is no cytotoxicity of MDS clones by LEN, suggesting an alternative method of erythropoiesis rescue. Although we know that LEN promotes the formation of signaling platforms, and recruitment of EpoR, we wished to determine whether there was an effect of LEN on EpoR expression, as EpoR expression is controlled through ubiquitination and proteasomal degradation. Treatment of erythroid progenitor cell lines and primary erythroid precursors with LEN increased cellular expression of Jak2-associated EpoR in a concentration dependent manner. There was no change in mRNA expression, supporting a post transcriptional mechanism. We then investigated whether receptor up-regulation was limited to EpoR, or included other cytokine receptors. We found that LEN induced expression of another Jak2 associated Type I receptor, IL3-R, but did not alter cellular expression of c-kit, a Type II cytokine receptor. Because Type I cytokine receptor turnover is regulated by a shared E3-ubiquitin ligase, and LEN inhibited both MDM2 and cereblon, we evaluated the effects of LEN on the E3-ubiquitin ligase, Ring Finger Protein-41 (RNF41), which regulates steady state or ligand independent, Jak2 associated Type I receptor internalization. We found that LEN inhibited the ubiquitination activity of RNF41, ultimately stabilizing EpoR membrane residence and increasing expression. In summary, MDS patients display ineffective hematopoiesis likely in part to decreased lipid raft assembly. Stimulation by Epo, or treatment by LEN, not only induced raft formation, but also induced the recruitment of both growth factor receptor, and downstream signaling intermediates into raft fractions to enhance EpoR signal fidelity. We have shown here two methods by which LEN may augment EpoR signaling. First, LEN increases lipid rafts and promotes recruitment of signaling effectors. Second, LEN increases and stabilizes the expression of EpoR through inhibition of the E3 ubiquitin ligase, RNF41. Therefore, we suggest here that LEN may have broad E3 ubiquitin ligase inhibitory effects. These data also indicate that lipid raft upregulation by LEN is mediated through GTPases, suggesting that GTPase activation may also occur via inhibition of specific E3 ubiquitin ligases, a question to be addressed in future studies.

Kyoto University (京都大学)
0048
新制・課程博士
博士(人間・環境学)
甲第16159号
人博第542号
新制||人||132(附属図書館)
22||人博||542(吉田南総合図書館)
28738
京都大学大学院人間・環境学研究科相関環境学専攻
(主査)教授 小松 賢志, 教授 五十嵐 樹彦, 教授 川本 卓男
学位規則第4条第1項該当

RNF11 (Ring Finger Protein 11) is an evolutionary conserved, 154 amino acid RING-H2 E3-ligase, containing different functional modules. The mature protein is anchored to membranes via a double acylation and localizes to early endosome and recycling compartments. Functional studies have implicated RNF11 in a variety of biological processes, including ubiquitylation, signal transduction and endocytic trafficking, but the molecular mechanisms underlying its function are still poorly understood. Combining affinity purification coupled with quantitative mass spectrometry and molecular biology techniques, we profiled the in vivo protein interaction network of RNF11 and revealed novel insight about its function. Specifically, we identified two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of GGA protein adaptors. We found that these motifs are the molecular determinants governing RNF11 sorting at the trans-Golgi network, as well as RNF11 internalization from the plasma membrane, and its intracellular trafficking between early and recycling endosome compartments. We also showed that RNF11, by recruiting the HECT E3-ligase Itch, regulates Itch catalytic activity and drives HECT-mediated ubiquitination of GGA protein family. As our preliminary evidence suggested that a similar mechanism could affect also other trafficking machinery proteins, such as the ESCRT-0 components, we proposed for RNF11 a central role in a finely regulated system where it acts both as an adaptor and a modulator of HECT-ligase activity, controlling the ubiquitination events underlying membrane traffic. Lastly, we demonstrated that a subgroup of Ubiquitin Binding Domain (UBD) - particularly UIM – interacts directly with RNF11, without requiring its ubiquitination, at odds with “conventional” UBD binding mode. This, in addition to identify the first specific binding partner or these UBD, open novel and intriguing perspective about RNF11 binding mode and mechanistic insights into the ubiquitin-network and the biological processes RNF11 is involved in.

The preimplantation mammalian embryo is sensitive to the environment in which they develop and grow, either in vivo or in vitro. Disturbance to embryo environmental conditions can affect embryo growth. Changes in gene expression is one of the embryo responses to such conditions that may influence developmental potential and phenotype during later gestation. The signalling network Myc/Max/Mxd function as a molecular switch that regulates cell growth, proliferation and differentiation by controlling a common set of genes. RING finger protein 17(Rnf17) enhance Myc activity by sequestering all four members of Mxd family and creating a "Mxd-null phenotype". The MXD family functions as MYC repressor proteins. Two isoforms were described for Rnf17, long (Rnf17L) and short (Rnf17S). Rnf17 is expressed in adult mouse testis and in the mouse preimplantation embryo. The Rnf17 proteins contain RING finger domain and varying copies of the Tudor repeat domain (Tdrd). The aim of my thesis was to study the potential for Rnf17 regulating the Myc/Max/Mxd network during preimplantation development on the phenotype of the blastocyst and foetal testes. First, F9 murine carcinoma cells were used to question whether Rnf17 can modulate Myc responses. Functional assays were developed for Rnf17 over-expression by expression plasmids or Rnf17 knockdown by RNAi construct. The specificity of Rnf17 E3 ligase was investigated by co-transfection of Flagg-tagged constructs for RNF17 and MXD1 in F9 cells. Second, expression of Rnf17 mRNA and subcellular localisation of RNF17 protein were examined in mouse preimplantation embryo and in E17.5 foetal testes under normal condition and in response to maternal protein diets (LPD; 9% casein, HPD; 30% casein and control NPD; 18% casein). Finally Rnf17 knockdown was performed in mouse embryo by siRNA microinjection to point out the role of Rnf17 in embryo development. RNF17 was able to degrade MXD1 by ubiquitinylation pathway in co-transfected Cos-1 cells. Rnf17-SV40 expression construct significantly induced Luciferase activity of c-Myc reporter construct (P≤0.05) when over-expressed in F9 cells, and knockdown of Rnf17 using Rnf17-RNAi construct significantly reduced Luciferase activity of Rnf17-sensor construct (P≤0.05). Rnf17 mRNA is expressed through preimplantation development, in E17.5 foetal testes and in F9 cells. Members of Myc/Max/Mxd are expressed in mouse preimplantation embryo. The expression pattern of RNF17 in F9 cells and mouse embryos was predominantly nuclear and also presented in the cytoplasm. RNF17 was only expressed in the nuclei of Sertoli cells with faint signal in prospermatogonia in E17.5 foetal testes. Knockdown of Rnf17 in microinjected embryos using Rnf17_siRNA reduced expression of Rnf17S, delayed blastocyst formation, and reduced RNF17 protein signal. Maternal diet differentially regulate expression of Rnf17 and c-Myc in mouse blastocyst and E17.5 testes. In conclusion, over-expression of Rnf17 can amplify Myc response in F9 cells. MXD is a target protein for Rnf17 E3 ligase. Knockdown of Rnf17 in mouse embryo delayed blastocyst development. This study implicates that Rnf17 involved in regulating Myc/Max/Mxd signalling network and indicates a role for Rnf17 in blastocyst proliferation and growth. Changes in Rnf17 expression have the capacity to modulate early embryonic development and may contribute to phenotypic changes occurring later in life.

Ring Finger Protein 10 (RNF10) è una proteina messaggero sinaptonucleare localizzata a livello delle sinapsi eccitatorie e associata con la subunità GluN2A del recettore NMDA. Stimolazione dei recettori NMDA contenenti la subunità GluN2A induce il trasporto di RNF10 dalla sinapsi al nucleo e la conseguente espressione di specifici geni bersaglio, alcuni dei quali svolgono un ruolo nella patogenesi della malattia di Alzheimer o nella modulazione della morfologia delle spine dendritiche. E’ inoltre importante osservare come l’espressione di RNF10 sia ridotta negli ippocampi di pazienti con malattia di Alzheimer e come oligomeri di Aβ siano in grado di indurre il trasporto di RNF10 dalla sinapsi al nucleo. Il principale obiettivo del progetto di dottorato è la caratterizzazione del ruolo di RNF10 nei processi cognitivi e la plasticità sinaptica utilizzando gli animali RNF10 KO quale modello sperimentale. I topi RNF10 KO sono caratterizzati da una riduzione del peso corporeo e da un concomitante aumento dell’assunzione di cibo in assenza di modificazioni significative dei livelli di glucosio, insulina e FGF-21. L’analisi molecolare ha mostrato una riduzione nell’animale RNF10 delle proteine coinvolte nel metabolismo di APP nell’ippocampo e una conseguente riduzione dei livelli di Aβ. L’analisi morfologica ha mostrato un aumento significativo delle spine dendritiche mushroom e una concomitante riduzione delle spine sottili. Una approfondita analisi del comportamento ha rilevato come gli animali RNF10 KO non presentino modificazioni del comportamento motorio, sia in animali adulti che anziani, mentre è stata osservata una moderata alterata alterazione sia dell’esplorazione che della memoria a lungo termine. Inoltre, gli animali RNF10 sono caratterizzati dalla completa assenza di potenziamento a lungo termine nell’area CA1 dell’ippocampo. In conclusione, questi risultati suggeriscono il coinvolgimento di RNF10 nella patogenesi della malattia di Alzheimer e indicano un possibile ruolo di questa proteina nelle alterazioni delle capacità cognitive associate all’invecchiamento.
RING Finger Protein 10 (RNF10) is a novel synapse-to-nucleus protein messenger, enriched at the excitatory synapses and associated with the GluN2A subunit of NMDA receptors. RNF10 translocates from synapses to the nucleus, upon the activation of synaptic GluN2A-containing NMDA receptors, inducing the expression of specific target genes that have a role in Alzheimer’s disease (AD) pathogenesis or that are associated with the regulation of dendritic spine morphology. Notably, RNF10 expression is reduced in AD patients' hippocampi at the earlier stages of the disease and Aβ oligomers trigger RNF10 translocation from the synapse to the nucleus. The main objective of the PhD project is to characterize the role of RNF10 in cognitive function and synaptic plasticity, using the RNF10 KO mice as an experimental model. RNF10 KO mice present reduced body weight with an increase in food intake, and no alterations in glucose, insulin or FGF-21 levels. The molecular characterization shows downregulation of the amyloid cascade players in the hippocampus, and a consequent decrease in amyloid levels. Concerning morphological analysis, RNF10 KO animals show a significant increase in mushroom-type dendritic spines and a decrease in thin-type spines. In the behavioral characterization, both adult and aged KO animals have normal locomotion, but displayed slightly altered exploratory behavior, as well as an impaired long-term memory function. Moreover, RNF10 KO animals present a complete loss of long-term potentiation in CA1 area of the hippocampus. Overall, these data suggest an involvement of RNF10 in AD pathogenesis, pointing towards a possible role for RNF10 in cognitive dysfunction along aging.

An active synapse-to-nucleus communication is essential for long-term changes in neurons, like the regulation of neuronal plasticity and shaping neuronal morphology. Next to the fast electrochemical signaling, neurons employ a slower mechanism that involves a recently discovered class of proteins, the synaptonuclear messengers. Different studies showed the pivotal role of synaptonuclear messengers in the modulation of synaptic transmission at excitatory synapses. On the other hand, alterations of synaptonuclear messengers’ activity have been correlated to synaptic failure as observed in different synaptopathies, including both neurodevelopmental disorders and neurodegenerative diseases. Ring Finger Protein 10 (RNF10) has been recently identified as a novel synapse-to-nucleus signaling protein that specifically links the activation of synaptic GluN2A-containing NMDA receptors (NMDARs) to gene expression. RNF10 synaptonuclear trafficking is responsible for the remodeling of dendritic spines that substance the postsynaptic modifications required for long-term potentiation (LTP). However, the molecular mechanisms leading to NMDAR/RNF10 complex disruption and for initiating the importin-mediated trafficking of RNF10 to the nucleus remain unclear. In this PhD project we investigated the molecular mechanisms that underlie RNF10 activation and in this matter we discovered a protein kinase C (PKC)-dependent phosphorylation event on RNF10-Ser31, which drives RNF10 synaptonuclear trafficking. Moreover, we show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31.Based on these results, we investigated the role of RNF10 in vivo, in the RNF10-/- mouse model. In particular we studied the putative involvement of the synaptonuclear protein in neurodevelopment, focusing our attention on the first three weeks of postnatal life, which represents the critical period for neuronal differentiation and synaptogenesis in rodents. We found that RNF10-/- mice have an alteration in brain morphology, in particular in the hippocampal area, and impaired cognition. At a microscopic level, RNF10-/- deficiency alters the molecular composition and the morphology of the glutamatergic synapse. In the CA1 region of the Hippocampus, dendritic arborization of RNF10-/- neurons is severely reduced and LTP induction is compromised. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture and regulating synaptic plasticity in hippocampal neurons.

Autosomal recessive leukodystrophy with ataxia and deafness (AR-LAD) is a rare white matter disease that slowly develops cerebellar ataxia and deafness. As disease progresses, the patients develop a fatal cardiomyophathy that strongly recalls that observed in patients affected by laminopathies, a group of disorders caused by mutations in A-type lamins (Lamin A/C), the major components of nuclear lamina. Linkage analysis and positional cloning experiments allowed us to identify the causative gene as RNF220 and two different homozygous missense mutations in highly conserved residues in exon 8 (c.1088G>A and c.1094G>A) (Dr. Bertini, unpublished data). RNF220 gene encodes for a RING finger ubiquitin ligase with a predicted molecular weight of 62.7 kDa highly expressed in liver, kidney and brain. Ubiquitin ligases catalyze the covalent attachment of ubiquitin to the substrate and play a key role in the ubiquitin-mediated proteolysis, however to date the role of RNF220 and the effects of pathological mutations associated with AR-LAD still need to be demonstrated. Our study has been focused on the functional characterization of RNF220, particularly we proposed to: 1) study the expression and subcellular distribution of RNF220, 2) verify if RNF220 may be involved in protein degradation via UPS and eventually determinate the spatial relationship between RNF220 and the different compartments associated with the proteasome, 3) investigate the involvement of RNF220 in the maintenance of nuclear lamina integrity, 4) investigate the possible interaction of RNF220 with molecules involved in the autophagic pathway. The results of this study have shown that: 1) RNF220 is an ubiquitin ligase with a nuclear distribution, 2) RNF220 co-localized with the 20S subunit, the catalytic core of the 26S proteasome, in nuclear speckles where is likely involved in protein degradation via the ubiquitin system, 3) RNF220 is a key player in the maintenance of nuclear integrity as shown by alteration/delocalization of Lamin A/C in AR-LAD fibroblast and in COS-1 cells ectopically expressing RNF220, 3) RNF220 forms a complex with Ambra1, a novel autophagic molecule, and co-localized with it in the nucleus, suggesting a RNF220 implication in autophagic pathway. Taken together, our results suggest that RNF220, the defective protein of AR-LAD is a key player of a novel degradation complex at nuclear level, connecting autophagic and proteosomal pathways and playing a crucial role in maintaining nuclear integrity.

Les gènes ING (Inhibitor of Growth) sont des gènes candidats suppresseurs de tumeurs conservés de la Levure à l'Homme. Les protéines ING ont des fonctions suppressives de tumeurs de type I ou « caretaker » car elles participent aux processus de maintien de la stabilité du génome en régulant la réplication et la réparation de l'ADN. Elles ont aussi des fonctions suppressives de tumeurs de type II ou « gatekeeper » puisqu'elles sont impliquées dans la régulation de la prolifération cellulaire de façon dépendante et indépendante de p53 et car elles contrôlent la transcription génique en participant au remodelage de la chromatine. L'objectif de ma thèse est de mieux comprendre l'implication de ING1, ING2 et ING3 dans les voies de suppression des tumeurs. Nos travaux montrent que ING1 est sumoylée sur la lysine 193 principalement par l'E3 SUMO ligase PIAS4, afin de réguler l'ancrage de ING1 sur le promoteur de gènes cibles pour réguler leur transcription. Nous avons aussi décrit pour la première fois l'implication de ING2 et de ING3 dans la réponse aux cassures double brin de l'ADN. Nous montrons que cette fonction est conservée entre ING2, ING3 et leur orthologues, respectivement, Pho23 et Yng2 chez la Levure Saccharomyces cerevisiae. ING2 contrôle l'accumulation de PIAS4 au niveau des sites de dommages et régule la sumoylation de l'E3 ubquitine ligase RNF168, afin de permettre la signalisation et la réparation des cassures double brin de l'ADN. ING3 est nécessaire à l'accumulation de 53BP1 et contrôle la réparation de ces dommages. Ces travaux contribuent donc à une meilleure connaissance du rôle des ING dans les voies de suppression des tumeurs. Ils permettent de mieux comprendre comment ING1 régule la transcription génique et décrivent une nouvelle fonction suppressive de tumeur de type I ou « caretaker » pour ING2 et ING3 dans le maintien de la stabilité du génome
ING (Inhibitor of Growth) genes are tumor suppressor gene candidates conserved from Yeast to Humans. ING proteins have type I tumor suppressive functions or "caretaker" because they participate in the maintenance of genome stability by regulating DNA replication and repair processes. They have also tumor suppressive functions of type II or "gatekeeper" because they are involved in the regulation of cell proliferation in p53 dependent and independent manners. They also participate in the regulation of gene transcription by regulating chromatin remodeling. The aim of my thesis was to better understand how ING1, ING2 and ING3 are involved in tumor suppressive pathways. Our work shows that ING1 is sumoylated on lysine 193 mainly by the SUMO E3 ligase PIAS4 to regulate ING1 anchoring on target gene promoters to control gene transcription. We have also described the involvement of ING2 and ING3 in the DNA double strand breaks response. We show the conservation of this function between ING2, ING3 and their orthologs, respectively, Pho23 and Yng2 in Yeast Saccharomyces cerevisiae. ING2 controls the accumulation of PIAS4 at DNA damage sites and regulates the sumoylation of the E3 ubiquitin ligase RNF168, to regulate DNA double strand break signaling and repair. ING3 is necessary for the accumulation of 53BP1 and promotes DNA damage repair. This work contributes to a better understanding of the role of ING proteins in tumor suppression. It thus provides new insights of how ING1 regulates gene transcription and emphasizes a new tumor suppressive function of type I or "caretaker" for ING2 and ING3 in the genome stability maintenance

The molecular pathogenesis of Alzheimer’s disease (AD) is still controversial, although genetic and cell biology findings indicate accumulation of Aβ, especially in soluble oligomeric conformation, as the driving force of synaptic dysfunction with concomitant activation of complex cascade of molecular events leading to dementia. In the last few years, several studies aimed at understanding how Aβ accumulation and assembly compromise synaptic structure and function of excitatory synapses. In this study, we evaluated how Aβ can affect the local and the long-distance trafficking, since the alteration of these mechanisms could represent a key determinant for synaptic dysfunction. We focused the attention on ADAM10, which activity prevents Aβ production. Notably the regulation of ADAM10 synaptic localization is neuronal activity-dependent, in particular LTP decrease, while LTD foster ADAM10 surface expression. Here we found that Aβ42 (500nM, 30 min) exposure results in an increase of ADAM10 synaptic availability. In particular, Aβ42 treatment leads to a decrease in the association between ADAM10 and AP2 complex, suggesting that the augment in ADAM10 synaptic localization is due to a decrease of the endocytosis. Interestingly, this mechanism is completely lost in the context of AD, suggesting that the increase in ADAM10 endocytosis, and thus the reduction of its activity towards APP, could be a synaptic mechanism of AD pathogenesis. In light of this consideration, we developed CPPs able to interfere with ADAM10 clathrin-mediated endocytosis and to restore the unbalance between exo- and endocytosis. This tool can be considered a potential disease-modifying strategy capable of modifying the progression of the disease and rescuing the pathological phenotype. Afterwards we describe a novel synapse-to-nucleus signaling pathway, involving the RNF10 protein, that specifically links activation of synaptic GluN2A-containing NMDARs to nuclear gene expression. In physiological conditions, RNF10 dissociates from the NMDAR complex in an activity-dependent manner and we provide compelling evidence for importin-dependent long-distance transport from synapto-dendritic compartments to the nucleus. These findings suggest that synaptonuclear trafficking of RNF10 is involved in the control of gene expression, which is necessary for synaptic plasticity in hippocampal neurons. Here we demonstrated that Aβ can affect RNF10 long-distance trafficking. In particular, during acute exposure, Aβ induces RNF10 translocation while in a chronic AD pathological context we found a reduction of the translocation, suggesting that RNF10 could be involved in AD pathogenesis.

Integral membrane proteins are targeted to discrete compartments through the action of a number of transport pathways. The post-translational modification of cargo with ubiquitin is a key regulator of protein sorting. Ubiquitinated cargo are bound by specific cargo sorting machinery and directed towards the appropriate destination. Therefore, the identification and characterization of the proteins involved in cargo ubiquitination is critical to understanding the regulation of protein sorting. In the work presented here, we characterize the role of the E3 ubiquitin ligases, RNF126 and Rabring7, in two distinct membrane trafficking pathways. First, we show that RNF126 and Rabring7 are involved in the ligand induced downregulation of cell surface receptors. RNF126 and Rabring7 associate with the EGFR, amongst other RTKs, and promotes its ubiquitination. RNF126 and Rabring7 are required for the efficient sorting of the EGFR through the late endocytic compartment. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. In addition, the depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. Second, we found that RNF126 regulates the sorting of the CI-MPR. In cells transiently depleted of RNF126, the CI-MPR is dispersed into a transferrin receptor positive endocytic compartment. This effect is specific to the CI-MPR as other cargos that are sorted between the endosome at the Golgi remain unaffected. We found that RNF126 physically associates with the clathrin adaptor GGA3 and promotes its ubiquitination, suggesting that RNF126 regulates GGA3 mediated CI-MPR sorting. Together, this work furthers our understanding regarding the role of ubiquitin in membrane traffic.

BACKGROUND: Moyamoya disease (MMD) is a chronic, occlusive cerebrovascular disease characterized by progressive stenosis of intracranial arteries, specifically the internal carotid arteries, and the compensatory formation of an abnormal vascular network at the base of the brain. The exact cause of MMD is still not well understood. Many factors including genetic, environmental, and immunologic have been associated with the disease. RNF213 is considered the main susceptibility gene, especially in Eastern Asian patients. The founder mutation, p.R4810K, has been associated strongly with MMD, especially in Japan and Korea but has been shown to have low penetrance and has never been described in non-Eastern Asian MMD cases. RNF213 encodes for an E3 ubiquitin-protein ligase with ATPase activity. It has described to regulate angiogenesis giving rise to the possibility that variants in RNF213 may play a role in cerebrovascular diseases other than MMD. OBJECTIVES: The aims of this study were to determine if variation in the RNF213 gene contributes to MMD in a cohort of 15 unrelated patients with MMD of predominantly European descent, to investigate other potential genes implicated in MMD in these 15 patients, and to investigate if RNF213 also influences more common vascular phenotypes. METHODS: Patient history, detailed family history and a blood sample were collected from 15 patients with well-characterized MMD. DNA was extracted from a peripheral venous blood sample, assessed for quality, and DNA concentration quantified by PicoGreen®. The extracted DNA was sent for whole exome sequencing. Genome_GPS_2.0 was used to carry out secondary analysis of sequencing data and all data were stored in Oracle TRC. The files were aligned using Novoalign, variants analyzed using GATK, visualized using IGV and annotated using BioR-Web. Variants of interest were determined using Ingenuity® Variant Analysis™. To determine if RNF213 also influences more common vascular phenotypes, previously collected and whole exome sequenced samples from the Mayo Clinic Florida Familial Cerebrovascular Diseases Registry were analyzed. Variants in RNF213 were determined using the same approach as for MMD. RESULTS: Likely pathogenic variants in RNF213 were found in 13% (2/15) of patients. The p.R4810K variant has been previously published as the founder mutation for MMD in Eastern Asian populations. The affected patient was also of Eastern Asian origin. The other variant, p.R4019C, was found in a European descent case and has been described as a candidate pathogenic variant. Eight variants in five other genes previously associated with MMD were found. Of these, one previously reported variant, p.D455H in RPTN, was found in two patients. The other seven have not previously been described in MMD. In the analysis of the potential role for RNF213 in other cerebrovascular diseases, 54% (22/41) of African American patients had a non-synonymous, exonic variant in RNF213 with a MAF of less than 3%. A novel RNF213 variant was also found in a Caucasian patient who had a subarachnoid hemorrhage. CONCLUSION: The p.R4810K variant in RNF213 was confirmed to only be associated with MMD in Eastern Asians and not found in other ethnicities. However, a variant in RNF213 was found in a Caucasian patient suggesting RNF213 may indeed be disease causing in patients of diverse origin. RNF213 may also be implicated in other cerebrovascular diseases suggesting a common pathogenesis while other genes also appear to be involved in the pathogenesis of MMD.

The Hereditary ataxias (HAs) are a group of heterogenous neurological disorders associated with multiple genetic etiologies and encompassing a wide spectrum of phenotypes, where ataxia is the prominent feature. HAs are characterized by degeneration of Purkinje cell and/or spinocerebellar connections, often associated with defects in additional brain structures, and all patterns of inheritance may occur. Similar to other fields of medical genetics, Next Generation Sequencing (NGS) has entered the HA scenario widening our genetic and clinical knowledge of this condition, but routine NGS applications still miss genetic diagnosis in about two third of patients. In this doctoral study, we applied multi-gene panels to define the molecular basis in 259 patients with a clinical diagnosis of HA and negative to tests for pathological expansion in SCA1, 2, 3, 6, 7, 8, 12, 17 and FXN. We found a positive molecular diagnosis in 25% of patients, whereas a similar number of patients had an uncertain diagnosis due to the presence of either variants of uncertain significance or lack of biological samples to determine segregation among family members. Hence despite a higher positive diagnostic rate compared to similar studies described in literature, a half of patients lacked any indication of the genetic cause of their disease. Using exome sequencing as a second-tier approach in some families, refractory to multi-gene panel analysis, did not significantly improved our diagnostic yield. On the other hand, NGS analysis in our cohort indicated that familial cases were more easily diagnosed rather than sporadic cases, and also that combining massive sequencing with detailed clinical information and family studies increases the likelihood to reach a molecular diagnosis. Among positive patients, we could expand clinical and allelic information in a subgroup of genes offering original description of new mutations and corroborating genetic findings with functional investigations that took advantage of different in vitro or in vivo platforms. In particular, through functional studies in SPG7 knock-down models of Drosophila melanogaster, we remarked that SPG7, whose mutations cause spastic paraplegia type 7, has a critical role in neurons more than in skeletal muscle. The high frequency of p.Ala510Val mutation in SPG7 observed in our cohort as well in similar studies performed elsewhere moved us to develop a humanized knock-in fruit fly model harboring that specific mutation and prepare preliminary characterizations. Similar studies in fruit fly were performed silencing AFG3L2, the gene causing SPAX5 in a child in association with an unusual, relatively milder phenotype. Furthermore, combination of skin fibroblasts and Saccharomyces cerevisiae as models was employed in the genetic characterization of new mutations in a novel recessive HARS-related phenotype whereas primary human cells, yeast and Danio rerio models were used to functionally characterize new HA-related mutations in COQ4. Finally, we could expand the clinical presentation of rare causes of HAs describing new dominant mutations in STUB1 and biallelic variants in RFN216, COQ8A, and ATP13A2. Altogether, studies performed during this doctoral work further underlined the usefulness of NGS in HAs and highlighted how NGS technologies rely on the integrated use of family and clinical studies and different in vitro/in vivo platforms to substantiate molecular findings. The latter platform will be also a tool for future investigations to dissect pathogenesis and to improve therapies.

碩士
國立陽明大學
微生物及免疫學研究所
107
Influenza A virus belongs to Orthomyxoviridae, and is a common pathogen to human. Influenza viruses cause yearly outbreak around the world, resulting in about three to five millions cases of sever illness. Because the virus mutates frequently, we have to predict vaccine strains every year, moreover preexisting immunity cannot provide protection fully. Mutations also render influenza viruses resistance to current antivirals. A better understanding of host-virus interaction may helps us identify potential target for new antiviral drugs. Activation of RIG-I-like Receptors ( RLR ) signaling pathway induces type I interferon production to defend against influenza infection. This signal is transmitted through the aggregation of the adaptor protein MAVS on mitochondria. Previously, we found that activation of RIG-I promoted protein degradation of influenza viral proteins. M2 and PB1-F2 were the most affected, so we try to understand the possible mechanism mediating their degradation. We have found that RNF125, an unbiquitin E3 ligase known to regulate RIG-I signaling negatively, played a role in RIG-I-induced PB1-F2 protein degradation. Here, we extended the study to M2. On the other hand, using different pharmacological inhibitors has previous helped us identified the autophagy may be part of RIG-I- induced viral protein degradation. In this study, we examined the role of autophagy in RNF125-mediated M2 degradation. First, we found the overexpression of RNF125 facilitated the protein degradation of M2. Consistently, knockdown of RNF125 reduced RIG-I-induced M2 degradation. Next, by using pharmacological inhibitors, we found the M2 expression could be partially rescued by autophagy inhibitor including 3-MA and Bafilomycin-A1, suggesting M2 might be degraded through autophagy upon RIG-I activation. Surprisingly, we didn’t observe the proteasome inhibitors, such as MG132 and lactacystin, can reduce degradation. Finally, we investigated that whether RNF125 affects influenza virus ( PR8 ) during infection. We found that in presence of overexpressed RNF125, M2 expression was reduced moderately. This may lead to the reduction of viral tirter we observed. We concluded that RNF125 may play a role in RIG-I-mediatied antivral activity through enhancing viral protein degradation.

By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer.
October 2016

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

碩士
國立中正大學
分子生物研究所
96
Over the past years, piscine nodavirus devastated the grouper culture industry in Taiwan and other countries and caused economic loss massively every year. Especially, larvae of orange-spotted grouper (Epinephelus coioides) were seriously infected by the Nervous Necrosis Virus (NNV). According to previous studies, Dr. Jimmy Kwang’s lab demonstrated that NNV coat protein localized in the nucleus and could induce apoptosis, suggesting that NNV coat protein was not only a structure protein but also involved in the gene regulation. Therefore, we tried to screen for the coat-associated proteins from the human fetal brain cDNA library by yeast two-hybrid assay. We obtained an interesting protein, human ring finger protein 2 (hRNF2). In comparison with the database of NCBI, we found that the cDNA of hRNF2 identified was not a wild-type form, but was an alternative splicing form with the last intron 5. We examined several human cell lines by RT-PCR and confirmed the existence of this alternative splicing RNA in cells. This cDNA encoded a C-terminus truncated protein (amino acid 1 to 306), which was named as hRNF2S. Surprisingly, the wild-type hRNF2 FL (amino acid 1 to 336) could not interact with NNV coat protein by yeast two-hybrid assay, but hRNF2S would. Furthermore, we used pull-down, co-immunoprecipitation assay and co-localization assay to confirm the interaction between coat protein and hRNF2S. We identify the RING domain of RNF2 as the region that is responsible for the interaction with coat protein. In addition, we successfully cloned the gene of orange-spotted grouper RNF2 (OsgRNF2) by degenerate PCR and 5’/3’ RACE. On the contrary to hRNF2 FL, OsgRNF2 FL could interact with coat protein weakly. C-terminus truncated OsgRNF 1-157 was sufficient to interact with coat. Since RNF2 was demonstrated to be a transcriptional repressor previously, we investigated the transcriptional repression ability of hRNF2 in the presence or absence of NNV coat protein. From preliminary data, we found that NNV coat protein enhanced the transcriptional repression ability of both hRNF2 FL and hRNF2S although coat protein itself did not show any transcriptional activation or repression ability.

碩士
國立中正大學
生命科學系暨分子生物研究所暨生物醫學研究
97
Viral nervous necrosis (VNN) is a worldwide disease among marine fishes and causes high mortalities at larval stage and considerable economic damage to the aquaculture industry. Nervous necrosis virus (NNV) is one of the major pathogens of VNN diseases and infects the larvae of orange-spotted grouper (Epinephelus coiodes) seriously. According to previous studies by Jimmy Kwang, the coat protein of NNV could enter the nucleus and trigger the process of apoptosis, implying that coat protein was not only a structural protein but also a potential protein involved in cellular genes regulation. To further study the functions of coat protein, yeast two-hybrid assay was used to screen for the NNV coat-associated proteins using the human fetal brain cDNA library. An alternative splicing form of human Ring finger protein 2 (hRNF2S) was identified. Surprisingly, NNV coat protein interacted with hRNF2S, but not with the full length hRNF2. In this thesis, yeast two-hybrid assay and GST-pull down assay were performed to further confirm the interaction and map the interaction domain between NNV coat and hRNF2 S / hRNF2 FL. NNV coat protein mainly interacts with the N-terminal region of hRNF2S, especially the RING finger domain of RNF2. Since NNV infects fishes naturally, the interaction between NNV coat and fish orange-spotted grouper (Osg) RNF2 was examined both in vitro and in vivo by yeast two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay and immunoflurorescence assay. Thus, the biological function of this interaction will be further studied in the future.

碩士
中國醫藥大學
生物科技學系碩士班
104
Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Our previous finding showed that extracellular signal-regulated kinases (ERK) inhibitor effective suppressed cardiomyocyte hypertrophy and apoptosis via inhibiting Angiotensin II (Ang II)-induced insulin-like growth factor II receptor (IGF-IIR) expression. As well, we discovered Ang II-induced heat shock transcription factor I phosphorylation (pHSF1) accumulated in cytosol, which is positive correlated with cardiomyocyte hypertrophy and apoptosis. However, the detailed mechanism by which ERK affects HSF-I phosphorylation to regulate IGF-IIR in heart cell remains elusive. In this study, we found that Ang II activated its downstream kinase ERK to increase IGF-IIR expression through its receptor, Angiotensin II type I receptor (AT1R). ERK activation subsequently phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase III (GSK3) on serine 303 in cardiac myoblast. Unexpectedly, we observed that Ang II-induced IGF-IIR expression was increased by ERK/GSK3 activation not though the DNA transcription, but via repressing IGF-IIR protein degradation. When the proteasome activity was inhibited, lots of ubiquitin-bound IGF-IIR complex was accumulated in cardiac myoblast, which is similar with the effect of Ang II. And we found that the E3 ubiquitin ligase of IGF-IIR, RING finger protein CXXVI (RNF126), was inhibited by p-HSF1 S303 to continually cause cardiomyocyte injury in hypertensive model. Afterwards, we confirmed direct effect of ERK/GSK3 inhibitor and compared with the widely used hypertension-Angiotensin II receptor blockers (ARBs) in vivo during 12 weeks Intraperitoneal(IP) injection into Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rat(SHR). Then we analyzed the cardiac function by echocardiography, observed the image of TUNEL, IHC, H&E staining on tissue sections, and isolated the left ventricular heart tissue to compare the target protein expression. Finally, we concluded that ERK/GSK3-activated pHSF1 at serine 303 and 307 repress the protein degradation of IGF-IIR by RNF126 decrease was accelerating cardiomyocyte hypertrophy and apoptosis during hypertension-induced HF. These findings provided us HSF1-influenced associated protein have therapeutic value in developing treatments for cardiac disease.