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Moon, Jennifer Kathryn. "Characterization of CUL1 and CUL2: Subunits of SCF E3 ubiquitin ligases in Arabidopsis thaliana." [Bloomington, Ind.] : Indiana University, 2004. http://wwwlib.umi.com/dissertations/fullcit/3162252.
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Loos, Trina Jane. "Determining the Function of Nuclear Bmp4." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2586.
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Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Madoz-Gurpide et al. recently observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF-β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. The results of chapter one report the existence of a nuclear variant of Bmp4. nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Chapter 2 and 3 further report that nBmp4 interacts with several subunits in the SCF E3 ubiquitin ligase, namely two Regulator of Cullins (ROC) proteins, five Cullin proteins, and two F-box proteins. Due to the known role of the SCF E3 ubiquitin ligase in regulating the cell cycle, the effect of nBmp4 on cell cycle progression was analyzed and the results show that nBmp4 affects the cell cycle by causing cells to accumulate in G0/G1. The association of nBmp4 and the SCF E3 ubiquitin ligase components and the affect that nBmp4 has on the cell cycle suggest that nBmp4 functions in the nucleus by inhibiting the SCF E3 ubiquitin ligase from ubiquitinating target proteins that are involved in regulating cell cycle progression. Finally, the initial stages in the generation of an nBmp4 over-expression mouse are described. The results of this research clearly change the traditional paradigm that Bmp4 performs all of its functions via extracellular signaling and introduce the existence of a nuclear variant that is involved in cell cycle regulation.
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Vadhvani, Mayur [Verfasser], Judith [Akademischer Betreuer] Stegmüller, Klaus-Armin [Akademischer Betreuer] Nave, and Till [Akademischer Betreuer] Marquardt. "The role of E3 ubiquitin ligase FBXO31-SCF in neuronal morphogenesis / Mayur Vadhvani. Gutachter: Judith Stegmüller ; Klaus-Armin Nave ; Till Marquardt. Betreuer: Judith Stegmüller." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2013. http://d-nb.info/1044047453/34.
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Brockelt, David [Verfasser], Judith [Akademischer Betreuer] [Gutachter] Stegmüller, and Tiago Fleming [Gutachter] Outeiro. "The role of the E3 ubiquitin ligase FBXO7-SCF in early-onset Parkinson's disease / David Brockelt. Betreuer: Judith Stegmüller. Gutachter: Judith Stegmüller ; Tiago Fleming Outeiro." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1112736522/34.
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El, Beji Imen. "Caractérisation biochimique et moléculaire du complexe SCF (SKP1-CULLIN-FBOX) chez le blé tendre." Phd thesis, Université Blaise Pascal - Clermont-Ferrand II, 2011. http://tel.archives-ouvertes.fr/tel-00999477.
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Les modifications post-traductionnelles des protéines constituent un niveau crucial de régulation de l'expression des gènes. Parmi elles, la conjugaison peptidique impliquant l'ubiquitine intervient entre autre dans la régulation de la stabilité protéique. La fixation de ce peptide de 76 acides aminés, extrêmement conservé, sous forme de chaîne de polyubiquitine, nécessite l'intervention de trois enzymes (E1, E2 et E3) et constitue un signal de dégradation de la protéine ainsi modifiée. Cette voie de régulation intervient dans de très nombreux processus biologiques. Les complexes SCF sont impliqués dans la voie de protéolyse ciblée. Ils représentent l' une des classes les plus fréquentes d'ubiquitine ligase E3 et ils sont composés de quatre sous-unités (Rbx, Cullin, SKP1, et F-box). La structure et la fonction des complexes SCF, ont été étudiées chez la levure, l'Homme et la plante modèle A. thaliana. Cependant, peu de travaux ont été réalisés chez des plantes cultivées, en particulier les céréales, telles que le blé. Cinq gènes codant pour la sous-unité Skp1 (TSK1, TSK3, TSK6, TSK11 et TSK16), cinq gènes codant pour la sous-unité F-box (ZTL, ATFBL5, EBF, TIR1 et ABA-T), un gène codant pour la sous-unité Cullin1 et un gène codant pour la protéine RBX du complexe SCF du blé, ont été isolés et clonés. Les différents tests d'interaction entre les quatre sous-unités du complexe SCF ont été réalisés par la méthode du double-hybride dans la levure en utilisant la technologie Gateway. Ces études ont montré que les deux protéines, TSK1 et TSK3, fixent spécifiquement différentes sous-unités F-box. Parallèlement, nous avons montré que la protéine TSK11 représente une structure particulière. Des études d'insertion/délétion sur la protéine TSK11 ont permis d'identifier un nouveau domaine indispensable à l'interaction. Les analyses par PCR semi-quantitative des différents gènes codant pour la sous-unité Skp1, dans trois tissus différents (feuille tige et racine), ont mis en évidence une expression constitutive des gènes TSK3, TSK6 et TSK11. Tandis que les gènes TSK1 et TSK16 sont exprimés préférentiellement dans les racines. Les analyses par PCR semi-quantitative sur des plantules de blé à différents stades de développement, ont mis en évidence une surexpression du gène TSK11 au moment de la floraison. Ce qui suggère que TSK11 est probablement un équivalent fonctionnel d'ASK1 chez Arabidopsis thaliana.
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Shang, Jinsai. "STRUCTURAL AND FUNCTIONAL STUDIES OF F-BOX-ONLY PROTEIN FBXO7 AND ITS INTERACTIONS WITH PROTEASOME INHIBITOR PI31." OpenSIUC, 2015. https://opensiuc.lib.siu.edu/dissertations/1053.
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F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.
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Nathan, James Alexander. "The RING-CH ubiquitin E3 ligase MARCH7." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612286.
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Cooper, S. E. "Studies of the E3 ubiquitin ligase Sina-Homologue." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597976.
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I have identified Sina-Homologue (SinaH) as a novel Drosophila protein that is homologous to the E3 ubiquitin ligase Sina, but has different expression patterns throughout development. In this thesis I investigate the biochemical mechanisms of SinaH directed degradation in cells, and the physiological role of SinaH in Drosophila. I show that SinaH can direct the degradation of the transcriptional repressor Tramtrack using two different mechanisms. One is similar to Sina and requires the adaptor Phyllopod (Phyl), and the other is a novel mechanism of recognition. This novel mode of targeting for degradation is specific for the Tramtrack isoform, Ttk69. Ttk69 contains a region that is required for binding of SinaH and for SinaH directed degradation. This region contains an AxVxP motif, which is the consensus sequence found in substrates of the mammalian Sina like proteins. These results suggest that degradation directed by SinaH is more similar to that found in higher eukaryotes. In order to identify novel SinaH substrates and potential adaptor proteins, a yeast 2-hybrid screen was carried out. As well as others, this identified Numb as a potential substrate, and the protein Bruce as an E2 ligase and adaptor molecule. GST pulldown assays and coimmunoprecipitation experiments were used to verify some of these interactors, and Numb was found to be directed for degradation in cells. Flies that were deficient in SinaH and in Sina and SinaH were created using homogolous recombination. The genetic data, cell degradation assays and expression profiles together suggest that Sina and SinaH have distinct functions.
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Van, den Boomen Dick Johannes Hendrikus. "Functional characterisation of the TRC8 E3 ubiquitin ligase." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609481.
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Chaugule, V. K. "Regulation of the ubiquitin RING E3 ligase Parkin." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1306179/.
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Post-translational modification of proteins by ubiquitin is a central regulatory process in all eukaryotic cells. Substrate selection and type of modification are events catalyzed by the E3 ligase, a component of the ubiquitin pathway. Several ubiquitin E3 ligases are implicated in cancer and other disease states, underlying the need for mechanistic insight of these enzymes. Parkinson’s disease is a neurodegenerative disorder characterised by the loss of dopaminergic neurons from the substantia nigra, the presence of Lewy Bodies, and pathogenic aggregates rich in ubiquitin. Autosomal Recessive Juvenile Parkinsonism (AR-JP), which is one of the most common familial forms of the disease, is directly linked to mutations in the Parkin gene (PARK2). Parkin is a RING E3 and catalyses a range of ubiquitination events (mono, multi mono, K48- and K63- linked poly) in concert with several E2s on a variety of substrates, including itself. Furthermore, Parkin is capable of binding the 26S proteasome and mediates selective degradation of target substrates. The data presented will demonstrate that the Ubiquitin-like domain (UblD) of Parkin functions to inhibit its auto-ubiquitination via a novel mechanism. Pathogenic Parkin mutations disrupt this inhibition and result in a constitutively active molecule. The inhibition is mediated by an intra-molecular interaction between UblD and the C-terminus of Parkin, and Lysine 48 on UblD participates in this interaction. The study also uncovered unique UblD/Ubiquitin Binding Regions (UBRs) on the C-terminus of Parkin that play a novel role in its RING E3 ligase activity. The observations provide critical mechanistic insights into the myriad functions of Parkin and the underlying basis of Parkinson’s disease.
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Cheng, Chi Ying. "Characterization of the adenovirus E4orf6/E1B55K E3 ubiquitin ligase." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103505.
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Human adenovirus type 5 (Ad5) has been engineered as therapeutic oncolytic viruses to selectively kill cancer cells. The earliest and most widely used is the Ad5 ONYX-015 virus which lacks the viral protein E1B55K. The adenoviral proteins E1B55K and E4orf6 were shown previously to be important for multiple functions during viral infection. Most of these functions are dependent on their formation of the E3 ligase complex with cellular proteins Cul5, Elongins B and C, and Rbx1. Therefore, in order to improve the current oncolytic adenovirus therapy, a better understanding on this E3 ligase complex is required. As the E4orf6/E1B55K E3 ligase contains components similar to other cullin E3 ligase complexes, the mechanism of the E3 ligase assembly was investigated. I showed that the E4orf6/E1B55K E3 ligase complex is formed in an unconventional way: E4orf6 uniquely contains three BC box motifs for its interaction with Elongin C unlike other cellular proteins, which contain only one BC box. In addition, the complex utilises neither of the two known mechanisms for recruiting Cul5. Ad5 is by far the best characterized of the more than fifty different adenovirus serotypes; however, it is unclear how representative its properties are with respect to all adenoviruses. Thus, the conservation of the E4orf6/E1B55K E3 ligase was studied systematically in members of other adenovirus subgroups. I demonstrated that the E4orf6 and E1B55K proteins from all serotypes can form an E3 ligase complex but with different cullin specificities: Ad4, Ad5, Ad9 and Ad34 recruit primarily Cul5, Ad12 and Ad40 recruit primarily Cul2, and Ad16 can recruit both. As for function, I showed that different serotypes degrade different ranges of substrates with the only common substrate to all being DNA ligase IV. I found clear evidence that E1B55K is the substrate binding component of the complex; however, I demonstrated that there is no correlation between binding and the capability to degrade specific substrates. These studies have shown clearly that considerable heterogeneity exists in the formation and function of the adenovirus E4orf6/E1B55K E3 ligase.L'adénovirus humain de type 5 (Ad5) a été modifié génétiquement à des fins thérapeutiques afin d'éliminer sélectivement les cellules cancéreuses. Le premier virus décrit et le plus communément utilisé est le Ad5 virus ONYX-015 dans lequel la protéine virale E1B55K est absente. Il a été précédemment démontré que les protéines adénovirales E1B55K et E4orf6 sont importantes pour de multiples fonctions lors de l'infection virale. La plupart de ces fonctions dépendent de la formation du complexe E3 ligase avec les protéines cellulaires Cul5, Elongine B et C, ainsi que Rbx1. Ainsi, une meilleure compréhension de ce complexe E3 ligase est nécessaire à l'amélioration des thérapies oncolytiques adénovirales actuelles. Puisque le complexe E4orf6/E1B55K E3 ligase contient des composants similaires à d'autres complexes culline E3 ligase, nous avons étudié le mécanisme d'assemblage de l'E3 ligase. J'ai ainsi démontré que le complexe E4orf6/E1B55K E3 ligase est formé de façon non conventionnelle : E4orf6 contient uniquement trois motifs BC pour son interaction avec l'Elongine C contrairement aux protéines cellulaires qui n'en contiennent qu'un. De plus, le complexe n'utilise aucun des deux mécanismes connus pour recruter Cul5. Ad5 est de loin le mieux caractérisé des plus de cinquante sérotypes différents d'adénovirus, pourtant on ne sait pas exactement à quel point ses propriétés sont représentatives des autres sérotypes, d'où l'importance de l'étude systématique de la conservation du complexe E4orf6/E1B55K E3 ligase parmi les membres des autres sous-groupes d'adénovirus. J'ai démontré que les protéines E4orf6 et E1B55K peuvent former un complexe E3 ligase dans tous les sérotypes, mais avec des cullines spécifiques différentes : les sérotypes viraux Ad4, Ad5, Ad9 et Ad34 recrutent principalement Cul5, Ad12 et Ad40 recrute principalement Cul2 alors que Ad16 recrute à la fois Cul5 et Cul2. En ce qui concerne les fonctions, j'ai démontré que différents sérotypes sont capables de dégrader des substrats différents, le seul substrat commun à tous les sérotypes étant la ligase d'ADN IV. J'ai mis en évidence qu'E1B55K est le membre du complexe qui se lie au substrat, mais il n'y a pas de correspondance entre la capacité de lier un substrat et celle de le dégrader. Ces expériences ont clairement démontré la grande hétérogénéité existant entre la formation et les fonctions du complexe adénoviral E4orf6/E1B55K E3 ligase.
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Dickens, Michael. "Small molecule inhibitors of Mdm2 E3 ubiquitin ligase activity." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/11960/.
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Half of cancers retain wild type p53 but have alterations in the pathways involved in p53 regulation. Murine double minute 2 (Mdm2) regulates p53 by acting as an E3 ubiquitin ligase, which tags p53 for degradation through the proteasome. A small molecule inhibitor, a 5-deazaflavin analogue, has previously been identified by high throughput screening to inhibit Mdm2 E3 ubiquitin ligase activity, thereby reactivating apoptotic function of p53 selectively in cancer cells. Ninety 5-deazaflavin analogues have been synthesised by an optimized existing method and a novel method of synthesis, using the required 6-anilinouracil and 2-p-toluenesulfonyloxybenzaldehyde.The biological ability of the 5-deazaflavin analogues to act as inhibitors of Mdm2 E3 ubiquitin ligase activity to reactivate p53 has been ascertained. A new quantitative biological assay was developed, by scientists based at the Beatson Institute, for 5-deazaflavin compounds, showing excellent inhibition of Mdm2 E3 ubiquitin ligase activity on the previous qualitative biological assay, to yield IC50 data. The biological results have established a clear and logical structure-activity relationship comprising of an electron-withdrawing hydrophobic substituent at the nine position and the N10 phenyl being a prerequisite for activity as a Mdm2 inhibitor. Also meta substitution of the N10 phenyl improves activity against Mdm2 E3 ubiquitin ligase activity. Hit optimization has occurred with 10-(3-chlorophenyl)-9-trifluoromethyl-5-deazaflavin being thirty times more active than the previous identified hit compound, 10-(4-chlorophenyl)-7-nitro-5-deazaflavin. Using the X-ray crystal structure of the Mdm2/MdmX heterodimer, an improved understanding of how Mdm2 acts as an E3 ubiquitin ligase is described and used to form a hypothesis of how 5-deazaflavin analogues function as inhibitors of Mdm2. The work suggests the principle that small molecular weight compounds can inhibit E3 ubiquitin ligases as a possible anti-cancer therapy, and provide the foundation and framework for additional studies and investigation in a new and developing field of medicinal chemistry.
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Menzies, Sam. "Ubiquitin E3 ligase mediated regulation of HMG-CoA Reductase." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/273763.
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Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.
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Lo, Shih-Ching. "Regulation of Nrf2 by a keap1-dependent E3 ubiquitin ligase." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4699.
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Thesis (Ph.D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 11, 2009) Includes bibliographical references.
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Zhang, Minghao. "Structural and functional studies of the CHIP E3 ubiquitin ligase." Thesis, Institute of Cancer Research (University Of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498516.
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Westerbeck, Jason William. "The SUMO-Targeted Ubiquitin Ligase Subunit Slx5 Functional Interacts with the SUMO E3 Ligase Siz1." W&M ScholarWorks, 2011. https://scholarworks.wm.edu/etd/1539626910.
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Peters, Sarah. "E3 ubiquitin ligase Hrd1 mediates the retrotranslocation of human Prion protein." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121158.
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Prion diseases are fatal neurodegenerative disorders. Ubiquitously expressed Prion protein (PrP) is found at the cell surface and is abundant in brain. However, a small proportion of PrP is found in the cytosol (CyPrP), arising from the ER-associated degradation (ERAD) pathway. CyPrP protects against Bcl2-associated X protein (Bax)-mediated apoptosis and most disease-causing PrP mutants (mPrP) completely or partially lose their ability to prevent Bax-mediated apoptosis through defective retrotranslocation of PrP (Jodoin et al., 2007). The E3 ubiquitin ligase HMG-CoA reductase degradation protein 1 (hrd1p) was found to mediate the retrotranslocation of human PrP in yeast (Apodaca et al., 2006). To determine the mechanism responsible for PrP retrotranslocation in the mammalian CNS, we either overexpressed an eYFP-Hrd1 fusion protein, or silenced endogenous Hrd1 in human CR7 glioblastoma cells and examined resultant levels of CyPrP. Hrd1 overexpression results in an increase in CyPrP, while, conversely, targeted knockdown of Hrd1 mRNA results in a marked decrease in CyPrP. Additionally we examined the effect of mPrP on the retrotranslocation of both PrP. Expression of mPrP substantially decreases CyPrP. Additionally, mPrP may be able to block the retrotranslocation of other model ERAD Hrd1 substrates, such as mutant transthyretin (TTRD18G), indicating the effect is not specific to PrP. The results show that E3 ligase Hrd1 mediates the retrotranslocation of cellular PrP and that mPrP fail to produce CyPrP by interrupting Hrd1-associated retrotranslocation machinery. This disruption also appears to interfere with the retrotranslocation of Hrd1-mediated ERAD substrate TTRD18G. We conclude that familial prion disease pathology could be due to both a loss of neuroprotective CyPrP and an inability to properly degrade other mutated or misfolded proteins through a disruption of the ERAD pathway. This accumulation of misfolded proteins over time, combined with general dysfunctions associated with aging, may explain delayed disease onset in individuals carrying pathogenic PrP mutations.Les maladies à Prion sont des maladies neurodégénératives mortelles. La protéine Prion (PrP), ubiquitairement exprimée, se retrouve en abondance dans le cerveau à la surface cellulaire. PrP provenant de la machinerie de dégradation des protéines associées au réticulum endoplasmique (ERAD) est localisée au cytosol (CyPrP). CyPrP protège contre l'apoptose induite par Bax. Aussi, des mutants de PrP (mPrP) causant les maladies ont perdu leurs capacité à prévenir l'apoptose causée par Protéine X associée à Bcl-2 (Bax), dû à une rétrotranslocation défectueuse de PrP (Jodoin et al., 2007). La E3 ubiquitine ligase appelée protéine HMG CoA réductase dégradation 1 (hrd1p) est un rétrotranslocateur de huPrP dans les levures (Apodaca et al., 2006). Pour déterminer le mécanisme responsable de la rétrotranslocation du PrP dans le SNC des mammifères, nous avons soit surexprimé ou soit diminué l'expression de Hrd1 dans les glioblastomes humaines CR7. La surexpression d'eYFP-Hrd1 entraîne une augmentation du CyPrP et la diminution de l'ARN messager de Hrd1 cause une nette diminution de CyPrP. De plus, nous avons étudié les effets des mPrP sur la rétrotranslocation de PrP. La surexpression de mPrP diminue considérablement la présence de CyPrP. Cette perturbation semble interférer avec la retrotranslocation d'un mutant de transthyrétine (TTRD18G), un autre substrat du ERAD médié par Hrd1. Ces résultats démontrent que la ligase Hrd1 est responsable de la rétrotranslocation de CyPrP et que les mPrP sont incapables de produire du CyPrP en bloquant la rétrotranslocation par Hrd1. Nous concluons que la pathologie des maladies familiales à Prion pourrait être due à une perte du CyPrP neuroprotecteur ainsi qu'à une perturbation de la machinerie ERAD. Par conséquent, une accumulation de protéines mal repliées, combinée à des dysfonctions générales associées au vieillissement, pourrait expliquer l'apparition et la progression lente de la maladie chez les individus portants les mutations pathogéniques de PrP.
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Patton, E. Elizabeth. "E3 ubiquitin protein ligase complexes that regulate G1-phase in budding yeast." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ59025.pdf.
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Williams, Jamie John Lewis. "Identification of substrates for the EPAC1-inducible E3 ubiquitin ligase component SOCS3." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/4013/.
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It is now accepted that there is a link between obesity and several diseases such as cardiovascular disease (CVD), diabetes, rheumatoid arthritis (RA), and atherosclerosis with the common initiating factor in pathogenesis being a state of low grade, chronic inflammation. This state, characterised by elevated levels of pro-inflammatory cytokines such as interleukin (IL) 6, leads to sustained activation of inflammatory signalling pathways such as the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and subsequently pathogenesis. Suppressor of cytokine signalling (SOCS) 3 is inducible by several stimuli including IL6 and 3'-5'-cyclic adenosine monophosphate (cAMP), and via these routes has been demonstrated to terminate IL6 signalling thus quenching JAK/STAT signalling and an inflammatory response. While SOCS3 was primarily characterised as a competitive inhibitor of intracellular signalling, it also functions as specificity factor for an elongin-cullin-SOCS (ECS)-type E3 ubiquitin ligase. In this role, it has been demonstrated to direct ubiquitin-mediated proteasomal degradation of several substrates and lysosomal routing. However, the full spectrum of SOCS3-dependently ubiquitinated substrates is unknown. Given that JAK/STAT signalling is critical in the development of chronic inflammatory disorders, delineating the role of SOCS3 as an E3 ligase might be therapeutically beneficial. However, given the broad range of SOCS3 stimuli, the availability of certain SOCS3 substrates might be conditional on the route of SOCS3 induction. Using a global proteomics approach, this study aimed to identify SOCS3-dependently ubiquitinated substrates in response to cAMP and thus elaborate on the already well-established role of cAMP in inflammation. Differentially stable isotope labelling of amino acids in cell culture (SILAC)-labelled, tandem affinity purified ubiquitinomes of wild type (WT) murine embryonic fibroblasts (MEFs) and SOCS3-/- MEFs, each expressing epitope-tagged forms of ubiquitin, were compared using mass spectrometry (MS) following cAMP-mediated SOCS3 induction. Using this approach, proteins modified by SOCS3 with the epitope-tagged form of ubiquitin should be enriched in WT MEFs but not SOCS3-/- MEFs. MaxQuant analysis of raw mass spectromeric data identified several candidate SOCS3 substrates. Of these, SOCS3 was found to interact with PTRF/cavin-1, a regulator of caveolae formation and stability. Other substrates were tested but with limited success. Co-immunoprecipitation studies showed that SOCS3 could precipitate cavin-1 however the interaction was reduced following the inhibition of protein tyrosine phosphatases (PTPs) using sodium orthovanadate and hydrogen peroxide. This was surprising since all known SOCS3 substrates are tyrosine-phosphorylated prior to interacting with SOCS3 via its Src-homology (SH) 2 domain. Consistent with this finding, SOCS3 did not interact with known cavin-1 tyrosine-phosphorylated peptides spotted on a peptide array. However, a full-length cavin-1 peptide array spotted with non-tyrosine-phosphorylated peptides showed specific interactions at multiple sites. It is proposed that this interaction might influence the localisation and stability of either protein. While SOCS3 was demonstrated to impact cavin-1 ubiquitination, the mechanism by which it does so or the functional consequence is still not clear. Immunoprecipitation of cavin-1 following the introduction of SOCS3 was accompanied by a shift in the polyubiquitin signal from a high molecular weight, seen with cavin-1 alone, to a low molecular weight. Furthermore, an enhanced K48-polyubiquitin signal was detectable in this low molecular weight fraction, which was focused around the molecular weight of cavin-1. It is not known if this ubiquitin signal is SOCS3-dependent. In conclusion, the project has identified and validated a novel substrate of SOCS3. However, the mechanism by which SOCS3 regulates cavin-1 ubiquitination or the biological function of the interaction is currently unknown.
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Depaux, Arnaud. "Régulation des complexes d'ubiquitinylation et de sumoylation par la ligase E3 hSIAH2." Paris 7, 2006. http://www.theses.fr/2006PA077094.
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Les modifications post-traductionnelles des protéines (phosphorylation, l'acétylation ou l'ubiquitinylation) permettent de réguler leur activité, stabilité, localisation ou interactions avec d'autres facteurs. Les complexes permettant la modification par l'ubiquitine ou Sumo bien que d'organisation similaire sont composés de protéines différentes : une ligase El qui active le résidu, une ligase E2 permettant le transfert de l'ubiquitine sur le substrat et une ligase E3 qui assure la spécificité de reconnaissance du substrat. Plusieurs familles de ligases E3 ont été décrites mais seule la famille de protéines à domaine RING Finger présente des membres impliqués dans les complexes de la sumoylation et de l'ubiquitinylation. Afin de caractériser de nouveaux partenaires des ligases à domaine RING Finger hSIAHl et hSIAH2 (human Seven In Absentia homolog), nous avons développé une expérience de double-hybride chez la levure en utilisant hSIAH2 pour appât. La caractérisation des partenaires ainsi isolés a fait l'objet de mon projet de thèse. J'ai mis en évidence des protéines impliquées dans l'ubiquitinylation (Ubiquitine, Ubc5 ou hSIAH) et la sumoylation (PIAS, SUMO et Ubc9). J'ai ainsi démontré que hSIAH2 est capable de former des homodimères et des hétérodimères avec hSIAH et que cette dimérisation permet de réguler la propre stabilité des deux protéines. D'autre part, j'ai montré que hSIAH2 catalyse l'ubiquitinylation de PIAS et sa dégradation par le protéasome. L'ensemble de ce travail a mis en évidence le rôle spécifique de hSIAH2 dans la régulation de la stabilité d'intermédiaires essentiels, à la fois, aux complexes d'ubiquitinylation et de sumoylationAfter synthesis, proteins are targeted to post-translational modifications such as acetylation, phosphorylation or ubiquitination. These mechanisms regulate their function, stability, localization or interaction with partners. Modification process by ubiquitin or sumo named ubiquitination or sumoylation respectively involve complexes with similar organization but compose of different enzymes. Their organization relies on Sumo or ubiquitin activating El enzyme, transferring E2-ligase and E3-ligase or sub-complex conferring the substrate specific récognition. El-ligase is unique for each complex, whereas E2 and E3-ligases are multiple. Among E3-ligase families, RING Finger protein family only has been involved in both modifications complexes. Two human homologs of Drosophila Seven In Absentia (hSIAHl et hSIAH2), belong to RING Finger E3-ligase family. In a yeast two hybrid assay, we have identified new SIAH interacting proteins. Their characterization has been the purpose of my PhD project. We have characterized partners implicated in both ubiquitination (ubiquitin, Ubc5 or hSIAH) and sumoylation (Sumo, Ubc9 and PIAS) pathways. In a first attempt, I have demonstrated that hSIAH proteins can form homo- or hetero-dimers. Dimerization régulates their stability via a proteasome dependent degradation. I have also demonstrated that hSIAH2 catalyzes the proteasome dependent degradation of PIAS1, a sumo E3-ligase. Altogether this study evidences an important rôle for hSIAH2 in the regulation of the stability of ubiquitination and sumolation complexes
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Mitchell, Jennifer Anne. "Characterization of Functional Domains of Cul3, an E3 Ubiquitin Ligase, Using Chimeric Analysis." PDXScholar, 2014. https://pdxscholar.library.pdx.edu/open_access_etds/1970.
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Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in concert with E2 enzymes to attach molecules of ubiquitin to protein substrates. There are seven CRLs in mammalian cells (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) that are highly homologous in sequence and structure. CRLs possess a highly conserved C- terminal domain that interacts with E2 enzymes, and a more variable N- terminal domain which recruits substrates through distinct substrate adapter molecules. Despite the structural similarity, these CRLs recognize distinct substrates and carry out unique functions in cells.
In order to characterize the functional domains of cullins that are responsible for their unique activity, we generated cullin chimeras for expression and analysis in mammalian cells. These chimeras are Cul3 mutants in which the C- terminal domain or N- terminal domain of Cul3 has been replaced by that of Cul1 or Cul2, respectively. These chimeras were cloned into a mammalian expression vector for the purpose of experimentation in cultured cells.
The chimeric cullin constructs provided a valuable tool for investigating how different functional domains of CRLs contribute to their specific functions in cells. In this study, we first investigated if the chimeras that we engineered were able to interact with their respective substrate adapters. We performed co- immunoprecipitation experiments in which we tested the ability of wild type, chimeric, or mutant cullin proteins to bind to three different substrate adapter proteins. We found that the chimera possessing the C- terminus of Cul1 and the N- terminus of Cul3 retains the ability to interact with the BTB substrate adapters Ctb57 and KLHL3. We also found that the chimera that possesses the C- terminus of Cul3 and the N- terminus of Cul1 was unable to interact with BTB proteins. Lastly, we found that the Cul1 adapter Skp1 was able to bind to Cul1, but did not bind to Cul3 or either chimera. We concluded that the chimera possessing the N- terminus of Cul3 likely retains the functional binding abilities of Cul3 at the N- terminus and would therefore be useful for conducting experiments.
In this study, we also used the cullin chimeras to investigate the binding interactions between E2 enzymes and cullin RING ligases. We performed co- immunoprecipitation assays to examine the interactions between E2 enzymes and wild type, mutant or chimeric cullin proteins. We found that E2 enzyme UbE2E1 selectively binds to Cul3 and not to Cull. Notably, the BTB binding region at the N- terminus of Cul3 is required for binding to UbE2E1. Furthermore, we found that UbE2E1 also binds to Cul3 substrate adapter protein Ctb57. These experiments revealed a novel interaction between and E2 enzyme and the N- terminus of Cul3, as well as with a Cul3 substrate adapter protein. In conclusion, the chimeras generated in this study have provided valuable information regarding what regions of CRLs are important for interactions with other proteins, and will continue to be a useful tool for investigating CRL structure and function.
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Ho, Meng-Hsuan. "CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF A COTTON RING-TYPE UBIQUITIN LIGASE (E3) GENE." MSSTATE, 2009. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11032009-165510/.
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A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.
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Li, Ningning. "FBXW7 (hCDC4) E3-ubiquitin ligase receptor : lineage potential and its commitment to cancer." Thesis, University of Nottingham, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595677.
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The crucial decision between stem cell self-renewal and differentiation must be tightly controlled, as it could otherwise contribute to tumorigenesis from expanding number of immortal, proliferative, partially differentiated cells. Despite the well-established links between signalling and transcriptional activity in relation to the regulatory mechanisms surrounding stem cell biology and tumorigenesis, little is known about how transcriptional activators/regulators are regulated after tumorigenesis is triggered. An obvious mechanism limiting the expression time of an activator is to destruct it via the ubiquitin-proteasome system (UPS).
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Maringer, Kevin. "Virion assembly of the herpes simplex virus type 1 E3 ubiquitin ligase ICP0." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/9147.
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Within the herpes simplex virus type 1 particle, a complex compartment termed the tegument is sandwiched between the nucleocapsid and envelope. Interactions between tegument components and the cytoplasmic tails of viral envelope glycoproteins are thought to play some role in the poorly defined process of tegument assembly. Here, virion incorporation of a major tegument protein, VP22, and the immediate-early transactivator of gene expression ICP0 was studied to improve our understanding of herpesvirus morphogenesis. In infected cells VP22 was found to bridge the viral glycoproteins gE and gM to form an assembly complex that also incorporates ICP0. VP22 is the major determinant for ICP0 packaging, although both gE and gM were shown to be required for optimal recruitment of ICP0 into the complex and virion. The glycoproteins are redundant for VP22, but not ICP0, assembly. A conserved region of VP22, and N-terminal sequences known to facilitate VP22 oligomerisation, contribute to complex formation. ICP0 domains important for VP22-ICP0 association and ICP0 assembly include the functionally essential RING finger motif and ICP0’s C-terminus. The immediate-early protein ICP27, known to be required for ICP0 assembly, was shown for the first time to be recruited into virions, independently of VP22 and ICP0. A mutant that packages enhanced levels of ICP0 exhibited augmented growth kinetics, implying that virion ICP0 performs important functions in infection. No impact of VP22 on ICP0 function could be determined, although a new role for VP22 in modulating gene expression was identified. Nevertheless, VP22 mutation modified ICP0 localisation in infected cells. Interestingly, ICP0 was shown to enhance late stages of virus replication. ICP0-mediated ubiquitination within putative cytoplasmic assembly domains was characterised to further investigate this late function. This work significantly improves our understanding of how glycoprotein-tegument interactions lead to the formation of multicomponent assemblies involved in herpesvirus morphogenesis.
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Iwamoto, Noriko. "The E3 ubiquitin ligase LNX1p80 downregulates claudins from tight junctions in MDCK cells." Kyoto University, 2009. http://hdl.handle.net/2433/124310.
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Cheng, Yen-Fu. "Role of the ubiquitin-proteasome pathway in the inner ear : identification of an E3 ubiquitin ligase for Atoh1." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/96458.
Full textAbstract:
Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 91-105).
Atoh1, the proneural basic-helix-loop-helix transcription factor, is critical for the differentiation of inner ear hair cells. Hair cells do not develop in mice that lack Atoh1, and overexpression of the transcription factor in embryonic ears induces differentiation of extra hair cells. The level of Atoh1 expression is under the control of a Wnt and Notch transcriptional regulatory network to keep the level of mRNA within a narrow range. Once the protein is made, it activates its own expression through an interaction with the Atoh1 enhancer, such that Atoh1 transcription is self-perpetuating. Because of this autoregulatory loop, halting transcription of the gene to maintain Atoh1 at an appropriate level would require that the amount of protein be decreased. Since the ubiquitin-proteasome pathway regulates catabolism of key regulatory proteins, we assessed its role in the degradation of Atoh1. E3 ubiquitin ligases confer substrate specificity to degradation of proteins by transferring a ubiquitin tag to a specific protein substrate. Using an immunoprecipitation/mass spectrometry screening approach, we identified Huwe1, a HECT domain E3 ubiquitin ligase, as an Atoh1 binding partner. We validated the binding between Atoh1 and Huwe1 through reciprocal co-immunoprecipitation and mass spectrometry. We found that Huwe1 promoted polyubiquitylation of Atoh1 through a lysine 48-linked polyubiquitin chain. Mutation at a catalytic cysteine within the HECT domain of Huwe1 reduced the polyubiquitylation. We also defined a motif in the C-terminus of Atoh1 responsible for interaction with Huwe1. Inhibition of proteasomal activity, as well as Huwe1 depletion, stabilized Atoh1 in the cochlea and resulted in generation of new hair cells in the newborn cochlea.
by Yen-Fu Cheng.
Ph. D.
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Crichton, Jennifer E. "The Role of the E3-ubiquitin Ligase Trim17 in the Mitochondrial Cell Death Pathway." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23715.
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The upregulation of apoptosis is a hallmark of several neurodegenerative disorders including ischemic stroke. In neurons, as in other cell types, Bax and tBid are critical regulators of the intrinsic pathway upstream of mitochondrial outer membrane permeabilization (MOMP) and caspase activation. The characterization of the molecular events that occur during the early stages is therefore extremely important from a therapeutic standpoint. Here I show that two independent genetic pilot screens looking for novel regulators of Bax activation identified a common hit in the E3 ubiquitin ligase Trim17. Knockdown of Trim17 was found to protect against tBid-induced death in primary cortical neurons and allowed for the maintenance of mitochondrial function and oxidative phosphorylation under this apoptotic stress. The RING-domain of Trim17 was found to interact with Opa1 in mouse brain extracts. Furthermore, Opa1 co-immunoprecipitated with exogenously expressed full-length Trim17 from HEK293 cells. Knockdown of Trim17 in neurons increased Opa1 protein levels under steady-state conditions. These results suggest that Trim17 regulates Bax-dependent apoptosis in neurons via the modulation of Opa1 levels.
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Sankaran, Saumya M. "A functional analysis of the mammalian E3 ubiquitin ligase WWP1 in a yeast model." Waltham, Mass. : Brandeis University, 2009. http://dcoll.brandeis.edu/handle/10192/23252.
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Kinsella, Elaine. "Investigation of the E3 ubiquitin ligase UBR5, a novel regulator of Hh gene expression." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/21106.
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The Hedgehog (Hh) signalling pathway is essential for embryogenesis and regulating cellular homeostasis in the adult, however much remains unknown about the molecular mechanisms that control ligand expression. In 2002, Lee et al. demonstrated that conditional mutation of the Drosophila hyperplastic discs gene (hyd) resulted in increased hh levels, suggesting that Hyd can act as a negative regulator of hh gene expression. Based on this evidence, the aim of this project was to investigate the hypothesis that UBR5, the murine homologue of Hyd, acts as a novel regulator of Hh gene expression in mammals. To investigate this hypothesis in vivo, I utilized the developing mouse limb as a model system that is highly sensitive to abnormal Hh expression. Morphological analysis of Ubr5 limb mutant embryos did not reveal an obvious phenotype, however quantitative analysis of Ihh gene expression and its downstream targets at E13.5 demonstrated a significant decrease in levels. In addition, changes in the expression of Runx2 and Msx2 were detected. Therefore, these data indicate that UBR5 can act as a positive regulator of Ihh expression, in addition to regulating other factors involved in chondrogenesis. The role for UBR5 as a positive regulator of Hh expression was also supported by in vitro investigations, demonstrating that UBR5 is required for Shh expression in mouse embryonic stem cells. Morphological analysis of adult Ubr5 limb mutant mice revealed the presence of significantly shorter long bones. These observations support previous reports that interference with IHH during early chondrogenesis can negatively affect long bone growth in the adult. Interestingly, adult Ubr5 limb mutant mice also possess osteophytes, a feature typically observed in osteoarthritis (OA), in addition to sites of ectopic mineralization (EM) near tendons of the knee and ankle. Based on these observations and evidence from the literature, I hypothesize that in addition to the role for UBR5 as a positive regulator of Ihh expression in the bone, UBR5 also plays a role in ligament/tendon development and/or maintenance, whereby its loss results in defective ligaments/tendons that are incapable of stabilizing the joints of the limb, culminating in joint deterioration, as observed in OA, in addition to EM. However, further investigation is required to determine whether this is also related to deregulated Ihh. These experiments suggest that Ubr5 limb mutant mice could provide a novel mouse model in the study of OA and prompt the investigation of the potential role for EDD, the human homologue of UBR5, in OA initiation and progression.
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Metzger, Thibaud. "Molecular mechanisms of PLK1 recognition by CUL3/KLHL22 E3-ubiquitin ligase controlling mitotic progression." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ010/document.
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L’ubiquitination est une modification post-traductionnelle impliquée dans de nombreux mécanismes cellulaires. L’E3-ubiquitine ligase CULLIN 3 (CUL3) est un régulateur essentiel de la progression mitotique, ubiquitinant d’importants régulateurs mitotiques et contrôlant leur localisation subcellulaire. Plus particulièrement, notre travail décrit le rôle de la nouvelle E31 ligase CUL3/KLHL22 dans la régulation de l’activité localisée de Polo-like kinase 1 (PLK1) et de ce fait dans l’établissement d’une progression mitotique précise. Néanmoins, les mécanismes moléculaires qui régissent la reconnaissance de son substrat par CUL3 demeurent inconnus. L’activité catalytique de PLK1 ne semble pas être nécessaire à son interaction avec KLHL22, mais aussi bien son domaine kinase que Polo-box (PBD) suffisent à co-purifier KLHL22. Des mutations au niveau du motif DFG, situé en amont du domaine kinase,et du tryptophane 414 au sein du PBD semblent influer sur la reconnaissance de KLHL22. Les résultats obtenus montrent les premières indications biochimiques du mode d’interaction du complexe CUL3/KLHL22/PLK1Ubiquitination is a post-translational modification involved in many cellular processes. The E3 ubiquitin-ligase based on CULLIN 3 protein (CUL3) is an essential regulator of mitotic division in human cells by ubiquitinating several important mitotic regulators and controlling their subcellular localization. In particular, our work described the role of novel CUL3/KLHL22 E3-ligase in regulation of localized activity of Polo-like kinase 1 (PLK1) and there by faithful mitotic progression. However, the molecular mechanisms of substrate recognition by CUL3 remain unknown. The catalytic activity of PLK1 may not be required for binding KLHL22 but both the kinase and the Polo-box domains are sufficient to co-purify KLHL22. Mutating the DFG motif within the kinase domain and the tryptophan 414 within the PBD influence the binding to KLHL22. These results provide first insights into molecular mechanisms of CUL3/KLHL22/PLK1complex
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Lari, Federica. "Resolution of proteotoxic stress in the endoplasmic reticulum by ubiquitin ligase complexes." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:871e0484-3de4-4d0d-8206-4af16a8b743e.
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The eukaryotic endoplasmic reticulum (ER) is a multifunctional organelle, primarily responsible for the folding and maturation of secretory proteins, as well as lipid metabolism, calcium homeostasis, ubiquitin-dependent signalling and cell fate decisions. ER-associated degradation (ERAD) oversees protein folding and delivers misfolded proteins for degradation by the proteasome via ubiquitin conjugation mediated by RING-type E3 ubiquitin ligases. An intact ERAD is crucial to cellular homeostasis, as unresolved protein imbalances cause ER stress that ultimately lead to apoptosis. The human ER accommodates at least 25 E3s, however our understanding is mostly limited to Hrd1 and AMFR/gp78, both of which have a defined function in ERAD. To understand the contribution of ER E3s to cellular and organelle homeostasis, this study used mass spectrometry of purified E3 complexes to identify cofactors and build interaction networks of ER-resident E3s. These findings will form the foundation for investigating the biological roles of these ubiquitin ligases. Transcriptional analysis highlighted the centrality of Hrd1 among all ER-resident E3s in response to protein misfolding in the ER. Additionally, the contribution of individual Hrd1 complex components to resolving proteotoxic stress was assessed using a misfolded antibody subunit (IgM heavy chain), rather than conventional pharmacological treatments. The ERAD components essential for substrate degradation and survival under proteotoxic stress were identified, highlighting the pivotal role of Hrd1, its cofactor SEL1L and the Derlin family members. Finally, it was demonstrated that autophagy induction in response to proteasome inhibition is key to relieve the burden of protein misfolding in the ER, as it sustained the survival of cells defective for ERAD. Importantly, this study proposes a potential involvement of Hrd1 in signalling from the ER to autophagy, suggesting potential crosstalk between the ERAD and autophagic pathways.
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Fu, Wei. "Regulation of FOXO stability and activity by MDM2 E3 ligase." [Tampa, Fla] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002222.
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Joch, Monica. "The synaptic PDZ protein PICK1 is a novel substrate for the E3 ubiquitin ligase parkin /." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98736.
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Mutations in the parkin gene result in early-onset, autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin (Ub) ligase that promotes the covalent attachment of Ub to specific substrate proteins that are targeted for proteasomal degradation. Here, we identify the synaptic PDZ protein PICK1 (Protein Interacting with C-Kinase 1) as a novel substrate for parkin-mediated ubiquitination. We establish that the PDZ domain of PICK1 interacts with the extreme C-terminus of parkin, which encodes a type II PDZ-binding motif. We demonstrate in vitro that parkin directly ubiquitinates wildtype PICK1 but not a PDZ domain mutant, PICK1 KD/AA. In cells, ubiquitination of PICK1 is enhanced by expression of wildtype parkin protein, but not by a ligase-inactive mutant, C431F. Interestingly, both in vitro and in cells, we found that parkin predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Polyubiquitination is known to target substrates for proteasomal degradation whereas monoubiquitination may regulate protein-protein interactions and intracellular trafficking. Consistent with monoubiquitination, PICK1 levels were not elevated by knockdown of parkin expression in PC12 cells or in parkin knockout mouse brain and, furthermore, we found no evidence for parkin-mediated proteasomal degradation of PICK1. Given that PICK1 is found in the same synaptic and postsynaptic density fractions as parkin and that PICK1 interacts with membrane proteins important for synaptic transmission, such as the AMPA receptor and dopamine transporter, ubiquitination of PICK1 by parkin offers a molecular mechanism for regulating synaptic function. These findings therefore provide important new insight into the role of parkin-dependent ubiquitination in synaptic transmission and Parkinson's disease.
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Nguyen, Huu Ngoc-Sa. "Characterisation of the expression and function of an E3 ubiquitin ligase, WWP1, in breast cancer." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492856.
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WWP1 is a ubiquitin ligase of the Nedd4-protein like family. Previous analysis in our laboratory suggested a possible relationship between WWP1 expression and breast cancer. This work attempted to characterise WWP1 expression and potential function in breast oncology. WWP1 mRNA expression was found to be up regulated mostly in ER+ breast tumour cell lines compared with non tumourigenic cell lines. In our experiments, WWP1 expression was not activated by oestrogen.
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Rutz, Natalja [Verfasser]. "Funktionelle Charakterisierung und zelluläre Interaktionen des putativen Cullin3-E3 Ubiquitin Ligase Substratadapters KCTD5 / Natalja Rutz." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1069290149/34.
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Soares, Pedro. "Small-molecule approaches to interrogate the druggability of the VHL E3 Cullin RING Ubiquitin Ligase." Thesis, University of Dundee, 2018. https://discovery.dundee.ac.uk/en/studentTheses/9a5ad7bc-e5e0-47d4-a4cd-29922f2ea9b0.
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Protein-protein interactions (PPIs) are prevalent in Nature and so are attractive targets for chemical biology and drug discovery. Modulating PPIs is challenging because of the nature of their interacting surfaces. Nevertheless, recent progress has led to the discovery of small molecule modulators of PPIs as chemical probes or lead compounds that have entered clinical trials. These findings have motivated more drug discovery programs to target PPIs. E3 ubiquitin ligases are attractive targets within the ubiquitin proteasome system, which function via PPIs. The von Hippel-Lindau protein (VHL) forms part of an E3 ubiquitin ligase for which the main biological function is to recognize and ubiquitinate the protein hypoxia inducible factor alpha subunit (HIF-α), its specific substrate, marking it for degradation by the proteasome. The VHL ligase is an important target for small drug development in two distinct approaches: in their own right, VHL inhibitors that block VHL catalytic activity could mimic a hypoxic response inside cell; moreover, VHL ligands can be conjugated into bifunctional degrader molecules (also known as PROTACs) to hijack VHL activity to induce targeted protein degradation. This work aimed to develop novel small molecules that target two different binding sites on the surface of VHL: 1) the hydroxyproline recognition site of HIF-α; and 2) a newly identified pocket on the VHL surface, and not involved in the VHL-HIF-α PPI. For the first binding site, I describe the structure-guided design and synthesis of a series inhibitors of the VHL:HIF-α PPI interaction, followed by their biophysical and cellular screening. These efforts led to the discovery of the first inhibitors of this interaction showing double-digit nanomolar affinities and good cellular activity. This work also led to the disclosure of compound VH298 as a potent and selective chemical probe of the hypoxia signalling pathway. Additionally, a series of thioamide containing analogues of VH298 were designed to probe the hydroxyproline recognition of VHL ligands. On the second VHL pocket starting from a weak-affinity fragment hit (Kd > 1 mM) I performed iterative cycles of synthesis, biophysical binding evaluation and fragment growing that yielded fragments with improved binding affinities to VHL. The most promising fragments achieved affinities around 100 µM to wild-type VHL. They also exhibited enhanced affinities for an R200W mutant, one of the most common mutations associated with the rare disease of Chuvash polycythemia. The developed efforts on this project disclose to the scientific community the first selective chemical probe of the hypoxia signalling pathway, and new ligands useful for the development of a new generation of PROTACs. The improved fragments targeting the second pocket could in future be developed into more potent and selective ligands targeting the disease-relevant mutation, and could also be used for novel PROTAC conjugation.
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Bulatov, Emil. "Interactions, assembly and fragment screening of the multisubunit SOCS2-EloBC-Cul5-Rbx2 E3 ubiquitin ligase." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708591.
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Vega-Sanchez, Miguel E. "The E3 ubiquitin ligase SPL11 regulates both programmed cell death and flowering time in rice." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1216996821.
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Pathe, Claudio. "Functional analysis of Shigella encoded IpaH E3 ubiquitin ligases in cell-autonomous immunity." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/285110.
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Shigella flexneri is a highly adapted pathogen that invades the host cytosol and causes bacillary dysentery. Shigella has evolved powerful countermeasures to disarm host defense mechanisms; amongst them a family of twelve bacterial E3 ubiquitin ligases (IpaH) that are structurally unrelated to eukaryotic enzymes. IpaH ligases are injected into the host cytosol via the bacterial type III secretion system (T3SS) to manipulate the host cell and counteract anti-bacterial defense pathways. My work demonstrated that IFN-induced guanylate-binding proteins (GBPs) are novel targets for IpaH9.8. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria unless they are ubiquitylated by IpaH9.8 and consequently degraded by the proteasome. IpaH9.8 targets GBP1, GBP2, and GBP4, thereby causing a transient poly-ubiquitin coat comprising K48 and K27-linked chains around S. flexneri, which leads to the proteasome-dependent destruction of existing GBP coats and the re-establishment of bacterial motility and cell-to-cell spread. So far, ubiquitylation of bacteria has mostly been associated with anti-bacterial autophagy or immune signaling. However, the ubiquitin coat assembled around intracellular Shigella by IpaH effectors, in particular IpaH9.8, serves a pro-bacterial function, the first observed so far. In addition, I characterized IpaH1.4 and IpaH2.5 for their ability to prevent NF-κB activation by targeting LUBAC. I found that IpaH1.4 specifically binds the LUBAC component HOIP and mediates its proteasomal degradation, thus abolishing linear ubiquitylation of bacteria and consecutive NF-κB activation via NEMO and autophagy induction via optineurin. Lastly, I identified novel potential ubiquitylation targets for IpaH effectors in human cells using a mass spectrometry-based approach. The resulting IpaH interactome presents the groundwork for further investigations and will help to identify potentially unknown cellular defense mechanisms that are antagonized by Shigella flexneri.
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Tam, Chun-yee. "A novel role of the E3 ubiquitin ligase as a transcription regulation in eukaryotic cell nucleus." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43278528.
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梁靄褳 and Oi-ning Leung. "Identification and characterization of E3 ubiquitin ligase SIAH1 as a regulatory target of microRNA-135a in HeLa cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40738589.
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Leung, Oi-ning. "Identification and characterization of E3 ubiquitin ligase SIAH1 as a regulatory target of microRNA-135a in HeLa cells." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40738589.
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Tam, Chun-yee, and 譚雋怡. "A novel role of the E3 ubiquitin ligase as a transcription regulation in eukaryotic cell nucleus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43278528.
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Rohde, Anna Maria [Verfasser]. "Molecular Characterization of the RING E3 Ubiquitin Ligase LIN41/TRIM71 in Early Neurogenesis / Anna Maria Rohde." Berlin : Freie Universität Berlin, 2014. http://d-nb.info/1053959583/34.
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Kellner, Vanessa [Verfasser]. "Understanding the function of the Rtt101 E3 ubiquitin ligase in response to replication stress / Vanessa Kellner." Mainz : Universitätsbibliothek Mainz, 2018. http://d-nb.info/116011191X/34.
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Saraswathy, Vishnu. "Identification d’un nouveau rôle de la E3-ubiquitin ligase Mindbomb1 dans la voie Polarité Cellulaire Planaire." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://theses.univ-cotedazur.fr/2019AZUR6000.
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La morphogenèse est le processus qui définit la forme d'un organisme (ou d'une partie d'un organisme) nécessaire à son bon fonctionnement. Au cours de l'embryogenèse, la morphogenèse d'un organe nécessite des processus incluant la division cellulaire, les mouvements cellulaires et la différenciation cellulaire. Cependant, on sait peu de choses sur la façon dont ces différents processus sont coordonnés au cours de la morphogenèse d'un organe. Au cours de ma thèse, j'ai étudié deux voies de signalisation cellulaire différentes qui régulent la morphogenèse au cours de l'embryogenèse du poisson zèbre. Mon étude a révélé que la voie de signalisation Notch et la voie PCP (Polarité Cellulaire Planaire) contrôlée par Mib1 régulent respectivement la morphogenèse du tube neural et l'extension de l'axe embryonnaire.Au cours de la première partie de ma thèse, j'ai étudié le rôle de la signalisation de Notch dans la morphogenèse du tube neural du poisson zèbre. La signalisation Notch a déjà été bien étudiée pour son rôle dans la régulation de la neurogenèse lors du développement du poisson zèbre. Cependant, on ne sait pas si et comment la signalisation Notch régule la morphogenèse du tube neural du poisson zèbre. L'épithélialisation et la c-division sont des événements importants au cours de la morphogenèse du tube neural du poisson zèbre. Nos résultats montrent que, en plus de synchroniser la spécification des cellules neuronales, la suppression de la neurogenèse induite par Notch est essentielle pour l’acquisition de l’architecture neuroépithéliale et pour la réalisation de c-division. Ainsi, la signalisation Notch permet de former la moelle épinière de poisson zèbre.Les observations de la première partie de ma thèse ont conduit à l'identification du rôle de Mindbomb1 (Mib1) dans la signalisation PCP. Mib1, une ligase ubiquitine-E3 nécessaire à l'activation de Notch, régule les mouvements d'extension convergence (CE) nécessaires à l'élongation de l'axe de l'embryon au cours de la gastrulation du poisson zèbre. De manière intéressante, nous avons montré que Mib1, indépendamment de sa fonction dans la signalisation Notch, agit dans la voie PCP pour réguler l’extension de l’axe de l’embryon. Dans la voie de la PCP, Mib1 agit comme une ligase ubiquitine-E3 et régule l'endocytose du composant de la PCP Ryk afin d'assurer la médiation de la CE lors de la gastrulation. Ainsi, notre étude a révélé que, indépendamment de son rôle dans la signalisation Delta / Notch, Mib1 est important pour la voie PCP lors de la gastrulation du poisson zèbreDuring my PhD, I studied two different cell signaling pathways that regulate morphogenesis during zebrafish development. I found that the Notch signaling pathway and Mib1 mediated Planar Cell Polarity (PCP) pathway regulate neural tube morphogenesis and embryonic axis extension respectively.During the first part of my PhD, I addressed the role of Notch signaling in zebrafish neural tube morphogenesis. Notch signaling has been well studied for its role in regulating neurogenesis during zebrafish development. However, whether and how it regulates morphogenesis of the zebrafish neural tube is unknown. Epithelialization and c-division are important events during zebrafish neural tube morphogenesis. Our findings show that, in addition to regulating the timing and identity of neuronal cell fate specification, Notch mediated suppression of neurogenesis is essential for the acquisition of polarized neuroepithelial tissue architecture and the execution specific morphogenetic movements called c-divisions, in order to properly shape the zebrafish spinal cord. Observations from the first part of my PhD led to the identification of the role of Mindbomb1(Mib1) in PCP signaling. Mib1, an E3-ubiquitin ligase required for Notch activation, regulates convergent extension (CE) movements during zebrafish gastrulation, that are required for the axis elongation of the embryo. Interestingly, I found that Mib1, independent of its function in Notch signaling, act in the PCP pathway to regulate axis extension. In the PCP pathway, Mib1 acts as an E3-ubiquitin ligase and regulates endocytosis of the PCP component Ryk to mediate CE during gastrulation. Thus, my study discovered that independent of its role in Delta/Notch signaling, Mib1 is important for the PCP pathway during zebrafish gastrulation
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Perez, Mockus Dago Jose Gantas. "Regulation of apical basal polarity and mesoderm invagination by the E3 ubiquitin ligase Neuralized in Drosophila." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066702/document.
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Les cellules épithéliales fournissent différentes fonctions biologiques: elles servent de barrière entre l'extérieur et l'intérieur d'un organisme et forment un continuum mécanique à travers les jonctions adhérentes qui les connectent. Au cours du développement, elles subissent des modifications extrêmes pour former l'embryon: elles changent de forme, modifient leur position relative ou perdent leur intégrité épithéliale. La plus part de ces changement se basent sur la modulation de l'actomyosine corticale et jonctionale, et sur la modulation des protéines qui définissent et maintiennent la polarité apico basale. Neuralized (Neur) est une E3 ubiquitine ligase qui est conservée des nématodes jusqu'aux mammifères. Elle a été découverte pour son rôle dans la régulation de la signalisation Delta/Nocth. Dans ce travail on décrit deux autres functions Notch-indépendantes de Neur dans le remodelage des épithéliums. En premier temps, on montre que Neur régule négativement la protéine apicale Crumbs à travers une isoforme de Stardust, ce qui permet le remodelage de l'intestin postérieur de la Drosophile et favorise la migration trans-epithéliale des cellules germinales primordiales. Puis, on présente que, pendant la gastrulation, Neur module la contractilité de l'actomyosine dans le mésoderme, et indirectement dans l'ectoderme, pour contrôler la formation du sillon ventralEpithelial cells serve many biological functions: they act as a barrier to separate the interior from the exterior, and form a mechanical continuum through the junctions that interconnect them. During development, they undergo dramatic changes to shape the embryo: they change their shape, modify their relative position or lose their epithelial integrity. Most of these changes rely on the modulation of cortical and junctional actomyosin, and the regulation of the proteins that define and maintain the epithelial apical/basal polarity. Neuralized (Neur) is an E3 ubiquitin ligase conserved from nematodes to mammals. It was first discovered for its role in the regulation of Delta/Notch signalling. Here we describe two Notch independent roles of Neur in epithelial remodelling. First, we show that Neur negatively regulates the apical protein Crumbs though a specific isoform of Stardust. This allows the remodelling of the drosophila posterior midgut and favours the trans-epithelial migration of the primordial germ cells. Finally, we present that Neur modulates actomyosin contractility in the mesoderm, and indirectly in the ectoderm, to control ventral furrow formation during gastrulation
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Moncrieff, Sophie. "Drosophila E3 ubiquitin ligase Hyperplastic Discs interacts with Shaggy and regulates morphogen signalling in the developing eye." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/15881.
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The expression of the Drosophila melanogaster morphogen Hedgehog (Hh) plays a key role in co-ordinating proliferation and differentiation during animal development. Tight spatial and temporal regulation of Hh expression is essential for its correct function in these essential processes. Both mis-expression of its mammalian orthologue Sonic Hedgehog (Shh) and aberrant stimulation of the associated signalling pathway occur in a wide range of human tumours. Although there is extensive knowledge of the signal transduction pathway that is activated in a Hh-stimulated cell, very little is known about pathways governing the expression of the morphogen itself. The Drosophila tumour suppressor protein Hyperplastic Discs (Hyd), an E3 ubiquitin ligase, negatively regulates hedgehog (hh) expression and Hh pathway activity by independent mechanisms in the developing Drosophila eye. Genetically generated hyd mutant clones in the eye mis-express hh and the transcriptional activator of Hh target genes, Cubitus interruptus (Ci), and cause overgrowth of the surrounding wildtype tissue. However, the underlying molecular mechanism(s) by which Hyd regulates these morphogen regulatory pathways is not known. Hyd may be involved in ubiquitylating target proteins in these pathways, which could have degradative or non-degradative outcomes. In order to elucidate Hyd’s molecular role in potential morphogen regulatory pathways, I applied a proteomics-based approach to identify novel Hyd binding partners and ubiquitylated substrates. Tandem affinity purification in combination with mass spectrometry was used to purify and identify Hyd and its complexed binding partners from Drosophila cells. Binding and ubiquitylation assays were subsequently used to verify and characterize the interactions. In addition, a biased, literature-guided approach was applied to identify likely Hyd binding partners based on their involvement in morphogen signalling and conservation across species. Finally, to assess the functional consequences of a newly identified interaction, I used a Drosophila in vivo model to determine whether the novel binding partner was capable of modifying the hyd mutant phenotype. For this purpose, the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique was used to generate hyd mutant clones in the developing larval eye, which were expressing transgenes resulting in either the over-expression or RNAi-mediated knockdown of the gene of interest. My results indicate that Hyd is involved in regulating both Hh and Wg morphogen signalling in the Drosophila eye, and that the molecular mechanism of action may, at least in part, involve the protein kinase Shaggy (Sgg). Hyd interacts with the Hh and Wg transcriptional activator proteins Ci and Armadillo, respectively, as well as the Sgg kinase. Sgg is a negative regulator of both the Hh and Wg pathways, and acts to direct the proteolytic processing or degradation of the transcriptional effectors of these morphogen pathways. Sgg and its mammalian orthologue GSK3β were ubiquitylated in vitro, and GSK3β ubiquitylation was negatively regulated by the mammalian homologue of Hyd, EDD. Knockdown of sgg in eye disc cells mutant for hyd resulted in a dramatic rescue of the overgrowth phenotype. Loss of hyd in clones located in the anterior region of the eye disc resulted in low levels of the full-length Hh transcriptional activator protein Ci. This effect was reversed completely as a result of sgg knockdown. Furthermore, loss of hyd in eye disc clones resulted in elevated Hh and Wg morphogen expression. Mis-expression of hh in hyd mutant clones was significantly reduced upon over-expression of a constitutively active Sgg kinase. Hence sgg appears to genetically act downstream of hyd to regulate hh gene expression and Ci expression. In summary my results identify Sgg as a novel regulator of hh gene expression, whose activity may be regulated by ubiquitylation, and which may be acting downstream of Hyd in a ubiquitin-regulated manner to control both hh gene expression and Hh pathway activity in the developing Drosophila eye. Hyd may also regulate Hh pathway activity by directly interacting with Ci and affecting its activity. The results also indicate that Hyd may be a master regulator of both Hh and Wg morphogen signalling during Drosophila development.
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Mukherjee, Progya. "In vitro reconstitution of the ubiquitylation and disassembly of the eukaryotic replisome." Thesis, University of Dundee, 2018. https://discovery.dundee.ac.uk/en/studentTheses/1ad1c2bd-1d21-4f9e-bf76-eaad19fcf2d6.
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Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the eukaryotic replication complex, has been shown to be extensively regulated by post translational modifications, during its assembly. Therefore, it is not inconceivable that the process to unload the replication complex would also be a conserved and regulated process. In 2014, our lab discovered that the CMG complex undergoes post-translational modification in the form of ubiquitylation on one of the subunits of CMG, leading to its disassembly from the chromatin. Though the main players in the disassembly of CMG were known, viz the E3 ligase SCFDia2 and segregase Cdc48, very little was known about the mechanism of CMG disassembly. In the process of learning more about the disassembly of the replicative helicase from chromatin, I reconstituted the ubiquitylation of CMG and thereafter the disassembly of CMG helicase in vitro. My work resulting in the reconstitution of CMG disassembly in vitro is the first example of the disassembly of a multi-subunit physiological substrate of Cdc48. Though CMG is ubiquitylated in yeast extracts in vitro, it does not lead to its disassembly and therefore led me to find conditions necessary for the efficient ubiquitylation of CMG. I have further shown that purifying the E3 ligase associated CMG can be efficiently ubiquitylated in a semi-reconstituted system consisting of purified factors, necessary for the ubiquitylation of substrate. I investigated whether this efficiently ubiquitylated CMG can be disassembled by purified Cdc48 and associated co-factor Ufd1/Npl4 in vitro and found that disassembly is dependent on K48 linked poly-ubiquitylation of CMG. I have found that the reconstituted poly-ubiquitylation of CMG is restricted to the Mcm7 subunit of CMG, recapitulating the ubiquitylation of CMG in vivo, and my data points out that there are multiple sites of ubiquitylation on Mcm7. Through this work, I have also found that ubiquitylated Mcm7 no longer associates with the rest of the CMG components after disassembly of CMG. My assays and findings, open the door towards dissecting the molecular mechanism of the disassembly of CMG in greater detail.
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Pilsl, Anna. "Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-153287.
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