LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.

LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.

Philosophiae Doctor - PhD / Retinoblastoma-binding protein 6 (RBBP6) is a multi-domain human protein known to play a role in mRNA splicing, cell cycle control and apoptosis. The protein interacts with tumour suppressor proteins p53 and pRb and recent studies have shown that it plays a role in the ubiquitination of p53 by interacting with Hdm2, the human homologue of mouse double minute protein 2 (Mdm2), in which the RING finger domain plays an essential role. Recently, RBBP6 has been shown to ubiquitinate the mRNA-associated proteinYB-1 through its RING finger domain, causing it to be degraded in the proteasome.RING (Really Interesting New Gene) fingers are small commonly-occurring domains of approximately 70 amino acids in length which coordinate two zinc ions in a cross-brace fashion.They are characterized by a conserved pattern of eight Cysteine or Histidine residues which are involved in coordinating the zinc ions. In terms of this conserved consensus, the RING finger from RBBP6 is expected to coordinate the zinc ions through eight Cysteine residues, making it a “C4C4” RING finger similar to those identified in transcription-associated proteins CNOT4(CCR4-NOT transcription complex, subunit 4) and p44 (interferon-induced protein 44). The amino acid sequence of the domain also shares many similarities with the U-box family of domains, which have an identical three-dimensional structure despite not requiring zinc ions in order to fold. This thesis reports the bacterial expression of a fragment containing the RING finger domain from human RBBP6, and determination of its structure using heteronuclear Nuclear Magnetic Resonance (NMR) spectroscopy. Preliminary NMR analysis of the fragment revealed that the domain was folded, but that it was preceded by an unstructured region at the N-terminus. A shortened fragment was therefore expressed and used for structural studies. Isotope-enriched protein samples were generated by growing bacteria in minimal media supplemented with 15NNH4Cl and 13C-glucose and purified using a combination of glutathione agarose affinity chromatography, anion exchange and size exclusion chromatography. A complete set of heteronuclear NMR data was collected at 600 MHz from which almost complete assignment of the backbone, side-chain and aromatic resonances was achieved. By exchange of Zn2+ with 113Cd2+ we managed to confirm that the domain binds two Zn2+ ions, and confirm that they are coordinated in the expected cross-brace manner. Structural data in the form of 2-Dimensional Nuclear Overhauser Enhancement Spectroscopy (2D-NOESY), 15N-separated NOESY and 13Cseparated NOESY spectra were recorded and used to determine the structure using restrained molecular dynamics on the Combined Assignment and Dynamics Algorithm for NMR Applications (CYANA) platform.As expected, the structure contains a triple-stranded β-sheet packing against an α-helix and two zinc-stabilized loops as found in all RING fingers. However, it also contains a C-terminal helix which packs against an N-terminal loop which is similar to that found in many U-box domains.A search using the DALI server revealed that the structure is most similar to the U-box from CHIP (C-terminus of Hsp70-interacting protein), an E3 ligase that cooperates with Hsp70 to degrade unfolded proteins that cannot be refolded. Using NMR we showed that the domain dimerizes with a KD of approximately 200 Μm, which means that it is dimeric at the concentrations used for NMR structure determination. Chemical shift analysis showed the dimerization interface to be very similar to that identified in U-box domains found in C-terminus of Hsp70 interacting proteins (CHIP).The structural similarities reported here between the RING finger from RBBP6 and the U-box family lead us to conclude that RBBP6 may, like CHIP, play a role in protein quality control.

Philosophiae Doctor - PhD / Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.

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,...

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La Neuropathie à Axones Géants (NAG) est une maladie neurodégénérative rare et fatale caractérisée par une détérioration du système nerveux central et périphérique, impliquant les fonctions motrices et sensorielles. La détérioration massive du système nerveux est accompagnée d'une désorganisation générale des Filaments Intermédiaires ce qui la différencie de nombreuses maladies neurodégénératives où seuls les neurofilaments(NFs) sont affectés. La protéine déficiente, la gigaxonine, est la sous-unité d'une ubiquitine ligase E3, responsable de la reconnaissance spécifique des substrats MAP1B, MAP1S et TBCB, seuls connus à ce jour.Dans le but d'étudier le rôle de la gigaxonine sur la survie neuronale, la désorganisation du cytosquelette et d'avoir un modèle animal suffisamment fort pour envisager des tests thérapeutiques, j'ai caractérisé un modèle murin de NAG. Pour ce faire, j'ai réalisé une étude comportementale des fonctions motrices et sensorielles ainsi qu'une étude histopathologique. Les souris NAG (129/SvJ) développent un phénotype moteur modéré dès 60 semaines alors que les souris NAG (C57BL/6) présentent un phénotype sensoriel dès 60 semaines. Les données histopathologiques ne présentent pas de mort neuronale mais les NFs sont sévèrement altérés. Les NFs sont plus abondant, leur diamètre est augmenté et leur orientation hétérogène, comme c'est observé chez les patients NAG.Nos résultats montrent que l'absence de gigaxonine induit un phénotype moteur et sensoriel modéré mais par contre reproduit la désorganisation massive des NFs observée chez les patients. Ce modèle va nous permettred'étudier le rôle de la gigaxonine, une ligase E3, sur l'organisation des NFs et ainsi comprendre les processus pathologiques impliqués dans d'autres maladies neurodégénératives caractérisée par une accumulation des NFs et un dysfonctionnement du système ubiquitine-protéasome comme les maladies d'Azheimer, de Parkinson etd'huntington ou la sclérose latérale amyotrophique. / Giant Axonal Neuropathy (GAN) is a rare and fatale neurodegenerative disorder characterized by a deterioration of the peripheral and central nervous system. The broad deterioration of the nervous system is accompanied with a general disorganization of the Intermediate Filaments which makes it different from other neurodegenerative disorders wherein only neurofilaments (NFs) are affected. The defective protein, gigaxonin, is the substrate adaptator of an E3 ubiquitin ligase, in charge of the specific recognition of MAP1B, MAP1S and TBCB. In order to study the role of gigaxonin on neuronal survival, the cytoskeleton disorganization and to have a relevant GAN animal model to evaluate efficacy of GAN treatments, I have characterized a GAN mouse model. I did a motor and sensory behavioural study and an histopathologic study. The GAN mice (129/SvJ) shown mild motordeficits starting at 60 weeks of age while sensory deficits were evidenced in C57BL/6 GAN mice. No apparent neurodegeneration was evidenced in GAN mice, but dysregulation of NFs was massive. NFs were more abundant, they shown the abnormal increased diameter and misorientation that are characteristics of the human pathology. Our results show that gigaxonin depletion induces mild motor and sensory deficits but recapitulates the severe NFs dysregulation seen in patients. Our model will allow us to study the role of the gigaxonin-E3 ligase in organizing NFs and understand the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of NFs and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases.

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

Le métabolisme des cellules immunitaires est assujetti à un contrôle étroit. L’inflammation, la présentation antigénique et l’expansion clonale sont des évènements qui demandent un changement rapide dans le métabolisme des cellules. Notamment, la glutamine est grandement sollicitée lors de la maturation des cellules dendritiques, les macrophages et les lymphocytes B et T pour son rôle dans la synthèse des protéines et son implication dans la phosphorylation oxydative. Au repos, les cellules présentatrices d’antigènes (CPAs) expriment l’ubiquitine ligase E3 MARCH1. MARCH1 est une protéine membranaire qui régule la réponse immunitaire en ubiquitinant, entre autres, le complexe majeur d’histocompatibilité II (CMH II) et CD86. Lors de l’activation des cellules immunitaires, son expression est réprimée, ce qui permet l’accumulation du CMH II et de CD86 sur leur membrane. Nous pensons que MARCH1 régule négativement le métabolisme des cellules immunitaires. Parmi les protéines membranaires sous le contrôle de MARCH1 pourraient se trouver des transporteurs de glutamine. La baisse rapide de MARCH1 serait nécessaire pour permettre aux cellules de modifier leur métabolisme en augmentant le transport de la glutamine. Dans le mémoire présent, nous nous sommes intéressés à l’interactôme de MARCH1. Afin de découvrir de nouvelles cibles de MARCH1, nous avons utilisé la méthode du BioID dans des cellules HEK293T. Le BioID est une méthode innovatrice permettant l’identification d’interactions interprotéines. La protéine de fusion BioID2 permet la biotinylation et l’isolation des protéines adjacentes in vivo. Ces essais de proximité nous ont permis d’identifier 41 cibles potentielles de MARCH1. Nous avons analysé l’expression de 13 de ces protéines par cytométrie en flux. Nos résultats démontrent que MARCH1 induit la dégradation de NKCC1, CD147 et SNAT2. L’expression de MARCH1 dans les cellules HEK293T engendre une diminution de SNAT2 en surface. S’il avère que MARCH1 régule le métabolisme de la glutamine dans les cellules immunitaires, il s’agirait alors d’un nouveau mécanisme par lequel cette ubiquitine ligase E3 module la réponse immunitaire. SNAT2 est nécessaire dans l’adaptation des cellules pour leurs besoins en glutamine. Nous discuterons du rôle que joue cette protéine dans l’adaptation du métabolisme et la glutaminolyse. / Immune cell metabolism is subjected to a tight control. Inflammation, antigen presentation and clonal expansion are all events that comes with a rapid change in metabolism. Glutamine is highly solicited during dendritic cells, macrophages and B and T lymphocytes maturation, due to its role in protein synthesis and oxidative phosphorylation. At steady-state, antigen presenting cells express the ubiquitin ligase E3 MARCH1. MARCH1 is a membrane protein involved in the immune response through major histocompatibility II and CD86 ubiquitination and degradation. During their activation, MARCH1 expression is repressed. This allows for accumulation of MHC II and CD86 on the cell surface, but other membrane-bound receptors and transporters are also increased during that time. Among those, proteins involved in glutamine transport are increased and thus help immune cells to adjust their intracellular nutrient pool for their new metabolic needs. We propose that MARCH1 negatively regulates immune cell metabolism through the regulation of nutrient transporters. The rapid stop in the transcription of MARCH1 induces an increase in receptors on the cytoplasmic membrane. Here, we aimed to identify the MARCH1 interactome. In order to identify new MARCH1 targets, we used the BioID proximity assays in HEK293T cells. BioID is an innovative method for the identification of protein interactions. BioID2 protein fusion can be used for in vivo biotinylation and isolation of promiscuous proteins. These proximity assays allowed us to identify 41 potential MARCH1 targets. We analyzed the expression of 13 of these proteins and found that 3 were affected by MARCH1 expression. Our results show that MARCH1 induces the degradation of NKCC1, CD147 and SNAT2. More specifically, MARCH1 expression in HEK293T induces the internalisation of the glutamine transporter SNAT2. If MARCH1 proves to regulate glutamine transport in immune cells, this would be a novel mechanism by which this ubiquitin ligase regulates adaptive immune system. Indeed, SNAT2 is required for the cellular adaption of amino acids during maturation, including glutamine. We will discuss the implications of MARCH1 as a metabolic switch and the role this would have in glutaminolysis and antigen presentation.

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