Dissertations / Theses on the topic 'Ubiquitin ligase'
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1
Fan, Jun. "Investigating the crosstalk between Nedd4 ubiquitin ligases and PIAS3 SUMO ligase." Thesis, Queen Mary, University of London, 2017. http://qmro.qmul.ac.uk/xmlui/handle/123456789/31791.
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Previously it has been shown that Rsp5p, a member of Nedd4 ubiquitin ligases in yeast, is modified by the ubiquitin-like protein SUMO and that this modification is performed by Siz1p, a member of PIAS SUMO ligases that are in turn substrates of Rsp5p-dependent ubiquitylation, thus defining a previously unidentified system of crosstalk between the ubiquitin and SUMO systems in yeast. This project aims to identify whether similar crosstalk pattern exists in human cells. In vitro ubiquitylation assays showed that some of the human Nedd4 family members (Nedd4.1, Nedd4.2, WWP1) are capable of ubiquitylating the human SUMO ligase PIAS3, while in contrast, Smurf2 does not appear to be able to modify this protein. This modification is partially WW-PY-motif-dependent as ubiquitylation level of PIAS3 mutants with altered PY motifs conducted by Nedd4.1 or Nedd4.2 was reduced, but not completely disrupted. Interestingly, in vitro SUMOylation assay revealed that Nedd4.1 is SUMOylated even in the absence of SUMO E3 ligases and an apparent interaction between the SUMO E2 (Ubc9) and Nedd4.1 was observed both in vitro and in vivo. I show that auto- SUMOylation of Nedd4.1 is accompanied with the formation of thioester-linked conjugates between Nedd4.1 and SUMO, but these do not involve cysteine residues (C867, C778, and C627) within the HECT domain itself and is not occurring at a predicted SUMOylation consensus site (K357). Furthermore, I have shown that Nedd4.1 and SUMO1/2 colocalize in HeLa cells, and that overexpression of epitope tagged Nedd4 and SUMO1/2, followed by denaturing pull-downs demonstrates that both Nedd4.1 and Nedd4.2 can be SUMOylated in vivo. Meanwhile, I have generated a SUMO trap based on SUMO interacting motifs (SIMs) and confirmed its ability of capturing SUMOylated proteins both in vivo and in vitro. Its use reveals that Nedd4 SUMO conjugates could be captured by SUMO trap when Nedd4 and SUMO were co-expressed in HeLa cells, again confirming Nedd4.1 as a substrate for SUMO1 or SUMO2. In conclusion, I show that SUMOylation of Nedd4.1 does exist in HeLa cells, and on the other hand, some of Nedd4 family members are responsible for PIAS3 ubiquitylation in vitro, providing evidence of a crosstalk between Nedd4 family of ubiquitin ligases and PIAS family of SUMO ligases in mammals.
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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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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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Bazirgan, Omar Al-Kasim. "Functional analysis of the ubiquitin ligase Hrd1p with the ubiquitin-conjugating enzyme Ubc7p." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3246079.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed March 9, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Wertz, Ingrid E. "Identification and characterization of novel ubiquitin ligase enzymes /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2004. http://uclibs.org/PID/11984.
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Harvey, Kieran F. "Functional characterisation of the ubiquitin-protein ligase, Nedd4." Title page, contents and abstract only, 2000. http://web4.library.adelaide.edu.au/theses/09PH/09phh3411.pdf.
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Addenda pasted into back end-paper. Bibliography: leaves 107-147. The studies presented in this thesis have identified Nedd4 as a key protein involved in Na+-dependent downregulation of the epithelial Na+ channel (ENaC). These studies have aided in the molecular understanding of the familial hypertensive disorder, Liddle's syndrome, and the regulation of blood pressure in general.
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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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Lee, Crystal Jayne. "Insights into the regulation of the CRL4D̳T̳L̳ ubiquitin ligase." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/70389.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.In title on title page, double-underscored "DTL" appears as superscript. Cataloged from PDF version of thesis.
Includes bibliographical references (p. 131-140).
The eukaryotic mitotic cell cycle is a strictly ordered process by which cells accurately duplicate their genome and divide into two. Ubiquitin-mediated degradation of key cell cycle regulators ensures that the cell cycle phases progress in a unidirectional and orderly manner. Cullin E3 ubiquitin ligases (CRLS) comprise a large family of multi-subunit complexes that selectively recruit substrates via a substrate receptor and facilitate substrate ubiquitination and degradation. The CRL4DTL (Cullin 4 RING ligase, in association with the substrate receptor DTL/Cdt2/RAMP) ubiquitin ligase has recently emerged as a key regulator of cell cycle progression and genome integrity. Identified substrates CRL4DTL play critical roles in S phase progression, replication, DNA repair processes, transcription, and chromatin regulation. CRL4DTL_ mediated targeting is restricted to S phase and after DNA damage through a PCNA-dependent mechanism. Recent studies have focused on elucidating the requirements within the substrates that dictate CRL4DTL-mediated degradation. The majority of identified substrates have a specialized PCNA interaction peptide motif (PIP box) that distinguishes the substrates from the stable PIP box containing proteins and couples interaction with chromatin-bound PCNA with CRL4DTL recruitment. Very few studies have explored the regulation of the substrate receptor DTL in the context of CRL4DTL ligase activity. DTL contains multiple WD40 repeats in the N-terminus that are very highly conserved and a less conserved C-terminus that may have important regulatory function. We characterize DTL regulation during the cell cycle: DTL itself is degraded in an ubiquitin-dependent manner and degradation is dictated by an unidentified C-terminal determinant. DTL is also phosphorylated in the C-terminus. Here, we present the first study to directly examine the contribution of the C-terminus to CRL4DTL ligase activity in the context of live cycling mammalian cells. We find that the DTL N-terminus can interact with substrates whether or not the substrates have bound PCNA. Importantly, we find that elements within the C-terminus are not required for CRL4DTL ligase assembly, substrate recognition, and substrate degradation during S phase and after DNA damage.
by Crystal Jayne Lee.
Ph.D.
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Furniss, James John. "Ubiquitin-ligase-mediated transcription initiation in cellular stress defences." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/22004.
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Accurate regulation of gene transcription is essential for organismal survival, and is orchestrated by myriad transcription factors and cofactors (TFs). Little is known about how the intrinsic activity of TFs is controlled. Recent work has indicated that the selective proteolysis of TFs provided by the ubiquitin-proteasome system (UPS) plays an important role in stimulating gene expression through a ‘destruction-activation’ mechanism, whereby the degradation of a ‘used’ TF is thought to stimulate further ‘fresh’ TF binding and reinitiate gene transcription. TFs are targeted to the proteasome via E3 ligases that mediate the addition of ubiquitin molecules to form a chain on the substrate TF. These polyubiquitin chains may be extended by E4 ligases, which recognize substrates with four or more ubiquitin molecules, amplifying substrate targeting to the proteasome. In plants the immune response to many pathogens is regulated by the hormone salicylic acid (SA), which operates through the transcriptional coactivator NPR1 to induce large scale changes in gene expression. Proteasome-mediated degradation of NPR1 appears to be required for the activation of its target genes. Mutation of the E3 ligase prevents ubiquitination of NPR1, leading its to stabilisation and suppression of transcription. Chapter 3 of this work identifies the first E4 ligase, UBE4, involved in NPR1 regulation. Mutation of UBE4 resulted in reduced capacity to polyubiquitinate substrates and stabilized NPR1. In contrast to E3 ligase mutants, however, mutant ube4 plants displayed increased NPR1 target gene expression. These results suggest that initial ubiquitination of NPR1 may stimulate its ability to initiate transcription and that subsequent ubiquitin chain elongation limits NPR1 activity by targeting it to the proteasome. Chapter 4 describes a ubiquitin-protein-ligase (UPL) which is both novel and crucial to the SA-mediated defence response. Mutation of this UPL leads a large reduction in total cellular polyubiquitinated proteins and was associated with strongly enhanced disease susceptibility. Gene expression profiling of upl mutants revealed an intimate connection between cellular polyubiquitination and appropriate activation of SA-responsive gene expression programmes. Destruction activation was first described in yeast and is required for the regulation of yeast amino acid synthesis TF GCN4. GCN4 requires proteasome-mediated degradation to induce genes involved in amino acid production. Chapter 5 investigates the role of two E4 ligases in GCN4 turnover. While one mutation had little effect of GCN4-mediated transcription a second increased basal transcriptional levels, suggesting that an E4 is required for the prevention of spurious GCN4-mediated transcription. In summary the work presented here describes cellular mechanisms by which global and substrate-specific polyubiqutination are vital to regulation of gene transcription.
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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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Glockzin, Sandra. "Identifizierung und Charakterisierung von potentiellen Interaktionspartnern der Ubiquitin-Protein-Ligase E6-AP." [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=968491162.
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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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Lotte, Romain. "Caractérisation des interactions moléculaires entre la GTPase Rac1 et son régulateur HACE1 : perspectives en infectiologie et en cancérologie." Thesis, Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4087/document.
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La GTPase Rac1 est une protéine de signalisation intracellulaire qui joue notamment un rôle clé dans la prolifération cellulaire. Notre laboratoire a montré que la toxine CNF1, produite par les Escherichia coli pathogènes, catalyse l’activation de Rac1. Nous avons également identifié le rôle de la E3 ubiquitine-ligase HACE1, un suppresseur de tumeur avéré, dans la régulation par ubiquitylation de Rac1 actif. S’il est prouvé que la forme activée de Rac1 est une cible d’HACE1, le mode d’interaction de ces deux protéines reste à définir ainsi que le rôle de ces interactions dans l’infection et le cancer. L’objectif de mon travail a été de caractériser les interactions moléculaires entre HACE1 et Rac1. Nous avons testé l’hypothèse que des mutations ponctuelles d’HACE1 identifiées dans les cancers pourraient interférer avec son interaction avec Rac1 et sa capacité de contrôle de la croissance cellulaire. J’ai ainsi pu mettre en évidence que 13 mutations somatiques d’HACE1 issues de tumeurs séquencées altèrent sa fonction de contrôle de la croissance cellulaire. De plus, l’étude de ces mutations nous a permis d’identifier un groupe d’acides aminés, situés sur les ankyrin-repeats 5 à 7 d’HACE1, qui contrôle l’interaction d’HACE1 avec Rac1 et de ce fait son ubiquitylation. Enfin dans cette étude nous précisons le rôle du domaine intermédiaire d’HACE1 (MID) dans la spécificité d’interaction de la ligase avec la forme active de Rac1. In fine, la caractérisation de mutants d’interaction entre HACE1 et Rac1 ainsi que l’effet de la toxine CNF1 sur cet axe de signalisation doit nous renseigner sur l’importance de cette voie de régulation dans le cancer et l’infectionThe small GTPase Rac1 plays a key role in various intracellular signaling pathways including cell proliferation. Our laboratory has shown that the CNF1 toxin, produced by pathogenic Escherichia coli, catalyzes the activation of Rac1. We also identified the role of the E3 ubiquitin-ligase HACE1, a tumor suppressor, in the regulation by ubiquitylation of active Rac1. If the activated form of Rac1 is proved to be a target of HACE1, the mode of interaction between these two proteins remains to be define as well as the role of these interactions in infection and cancer. The aim of my work was to characterize the molecular interactions between HACE1 and Rac1. We tested the hypothesis that HACE1 point mutations identified in cancers could interfere with its interaction with Rac1 and its ability to control cell growth. We showed that 13 cancer-associated somatic mutations of HACE1, led to a defective control of cell proliferation. Moreover, the study of these mutations allowed us to identify a group of amino acids, located on the ankyrin-repeats 5 to 7 of HACE1, which controls the interaction of HACE1 with Rac1 and thus its ubiquitylation. We also identified a role for the intermediate domain of HACE1 (MID) in conferring the specificity of association of HACE1 to the active form of Rac1. Ultimately, the characterization of interaction mutants between HACE1 and Rac1 as well as the effect of the CNF1 toxin on this signaling axis will give us more insight on this regulatory pathway in cancer and infection
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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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Dai, Qian Patterson Cam. "The cochaperone and ubiquitin ligase CHIP in protein quality control." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,285.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology." Discipline: Pharmacology; Department/School: Medicine.
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St-Pierre, Pascal. "Functional roles of ubiquitin ligase gp78 in endoplasmic reticulum domains." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43202.
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The ubiquitin ligase gp78, involved in ER-associated degradation, has been extensively studied using the 3F3A monoclonal antibody, and correlates with increased metastasis incidence in cancer patients (review in Chiu et al. 2008) The 3F3A-labeled ER characterized as an ER domain in close association with mitochondria distinct of the reticulon-labeled ER tubules, the juxtanuclear and the perinuclear ER. Overexpression of gp78 saw 3F3A expansion to the peripheral ER, but remained in association with mitochondria. This association is calcium-sensitive as elevation in cytosolic calcium levels disrupted contact with mitochondria. Similarly, induction of calcium released from the ER through thapsigargin or ATP stimulation of purinegic receptors promoted dissociation of 3F3A labeled ER and mitochondria. Upon ER-mitochondria dissociation, the IP3R-labeled ER dissociated in a distinct domain from the 3F3A-labeled ER, indicating that regulation of the ER-mitochondria association by free cytosolic calcium is a characteristic of smooth ER domains and that multiple mechanisms regulate the interactions between these organelles.
Upon overexpression of gp78, the 3F3A labeled peripheral ER was identified as the site of gp78-mediated ubiquitylation. Gp78 polyubiquitylated substrates where stabilized through Cue domain interaction in the peripheral ER. Derlin-1 and derlin-2, involved in retrotranslocation of ERAD substrates, localized to a juxtanuclear ER domain, where ubiquitylated proteins accumulate upon proteasome inhibition. This segregation was shown in a non-overexpressing system using HT-1080 fibrosarcoma cells expressing elevated levels of endogenous gp78. This shows the special segraqgation of ERAD by gp78-mediatedubiquitin ligase activity in the peripheral ER. This may function in ER quality control and regulate the expression of smooth ER-associated substrates in response to physiological change.
Finally, we found that stimulation of gp78 at the cell surface by its ligand AMF disrupts ER-mitochondria interaction, calcium coupling and decreases gp78 ubiquitin ligase activity. Disruption of the ubiquitin ligase activity via mutation of gp78 RING domain or expression of ubiquitin mutant unable to form elongated polyubiquitin chains disrupts ER-mitochondria coupling. The increased fragmentation and reduced mobility of mitochondria upon gp78 overexpression were dependant on ubiquitin ligase activity and prevented by AMF treatment. These results describe a novel mechanism regulating mitochondrial dynamics via the ER ubiquitin ligase gp78.
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Mortensen, Franziska [Verfasser]. "Regulation and function of the ubiquitin ligase E6AP / Franziska Mortensen." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1138566217/34.
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Liu, Zhiqian 1974. "E3HistoneLASU1, a 500 kDa novel multi-functional ubiquitin protein ligase." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103171.
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During spermatogenesis histones must be degraded in late round and early elongating spermatids to permit chromatin condensation. Ubiquitin conjugation is activated and histones are ubiquitinated at this stage, suggesting that histone degradation may be mediated by ubiquitination. The activation of ubiquitin conjugation during spermatogenesis is dependent on the ubiquitin conjugating enzyme (E2) UBC4. We therefore studied whether histones are ubiquitinated by a UBC4 dependent ubiquitin protein ligase (E3) during spermatogenesis. E3Histone was identified by a biochemical screen and purified to near homogeneity. Mass spectrometry identified E3Histone as LASU1, a 482 kDa HECT domain protein and E3Histone conjugates ubiquitin to all core histories in vitro. UBC4-1 and UBC4-testis were the preferred E2s for E3Histone-dependent ubiquitination of histones. E3Histone was the major UBC4-1 dependent histone ubiquitinating E3 in testis. Anti-LASU1 antibody immunodepleted E3 Histone activity. Immunohistochemistry showed that E3Histone /LASU1 was predominantly expressed in nuclei from spermatogonia to mid-pachytene cells, but not detectable in spermatids. Histones are also ubiquitinated in spermatocytes. E3Histone/LASU1 was widely expressed in different mouse tissues. It was mainly expressed in the cytoplasm in most tissues, except in neurons of the brain and in early germ cells of the testis where it was expressed in the nucleus. In most tissues, E3Histone/LASU1 was expressed in epithelia. The wide expression of E3Histone/LASU1 suggests the existence of substrates of this E3 other than histones. Indeed, our assays showed that in vitro purified E3Histone stimulates polyubiquitination of Mcl-1, a BH3 region containing antiapoptotic protein. E3Histone may therefore regulate cell apoptosis by mediating degradation of Mcl-1. Since E3Histone/LASU1 was found previously to affect gene transcription and histone monoubiquitination is known to regulate gene transcription, we also evaluated the role of E3 Histone/LASU1 in histone ubiquitination in somatic cells. Depletion of E3Histone/LASU1 protein by siRNA did not affect the levels of free or ubiquitinated histories. In summary, E3Histone /LASU1 is a novel multi-functional protein that may mediate histone ubiquitination during meiosis and may be involved in apoptosis by triggering Mcl-1 degradation. Its wide expression and large non-catalytic region indicate that there are likely many other substrates of E3Histone/LASU1.
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Major, Sarah A. "Structural studies of HsCks1 and the SCF'S'k'p² ubiquitin ligase complex." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426387.
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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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Kohlmann, Sonja. "Die HECT-Ligase Hul5, eine neue Komponente der ER-assoziierten Proteindegradation." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-33597.
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Christensen, Devin Eugene. "Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9222.
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D'silva, Michael Aloysius. "Characterization of the ubiquitin protein ligase E6-AP by RNA interference." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=979007968.
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Plans, Calafell Vanessa. "Studies on RNF8, a ubiquitin ligase with a RING finger domain." Doctoral thesis, Universitat de Barcelona, 2005. http://hdl.handle.net/10803/1868.
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La ubiquitinació és una modificació postraduccional de proteïnes empleada en la regulació de molts tipus diferents de senyalitzacions cel·lulars i processos biològics. Els enzims responsables d'aquesta modificació postraduccional són les E1 o enzims d'activació de ubiquitines, les E2 o enzims de conjugació de ubiquitines i les E3 o lligases de ubiquitines i poden conjugar una o més ubiquitines sobre la proteïna substrat.L'heterodimer format per UBC13-UEV te activitat de conjugació de ubiquitines mitjançant enllaços que utilitzen la lisina en posició 63 de la ubiquitina en contes de la lisina en posició 48. Aquesta modificació no es reconeguda pel proteasoma i per tant no implica una degradació de la proteïna que la porta. La cerca de proteïnes que interactuen amb UBC13 ens va permetre de identificar dues proteines: RNF8 i KIAA0675 que contenen un domini RING finger, pel qual interactuen amb UBC13. RNF8 te activitat autocatalítica de lligasa de ubiquitines, el que li permet elongar cadenes de poliubiquitines tant per lisina 48 com per lisina 63; aquesta darrera depèn de l'activitat catalítica de UBC13. A més, RNF8 co-localitza amb UBC13 en estructures nuclears.
L'estructura en dominis d'RNF8 és la mateixa que la del regulador del checkpoint mitòtic CHFR, ambdues proteïnes tenen un domini RING finger de reclutament d'enzims de conjugació de ubiquitines, al extrem amino terminal i un domini FHA de reconeixement de fosfopèptids. RNF8 localitza en el nucli cel·lular i te un patró d'expressió depenent de cicle amb uns nivells proteics que augmenten progressivament de G1 a M, assolint un màxim en metafase seguit d'una brusca desaparició en anafase i una recuperació de l'expressió de la proteïna en fases més tardanes de la mitosi. La sobre-expressió de RNF8 causa una reducció de la població en G2-M i una acumulació de cèl·lules en G1. La depleció d'RNF8 mitjançant la tècnica de RNAi no causa cap efecte significatiu sobre el cicle cel·lular basal. En canvi, la depleció d'RNF8 causa un retard en la sortida de mitosi després d'un arrest induït per nocodazole, fet que s'associa amb una reducció del turnover de la ciclina B1, que és un substrat de l'APC/C. Recíprocament, la sobre-expressió de RNF8 impedeix a les cèl·lules d'acumular-se en G2-M en resposta a un tractament amb nocodazole. A més l'activació del Spindle Assembly Checkpoint (SAC), mitjançant un breu tractament amb nocodazole, en combinació amb la sobre-expressió d'RNF8 impedeix que la proteïna del checkpoint Mad2 es localitzi als quinetocors. Aquesta funció d'RNF8 depèn de l'activitat lligasa ja que un mutant d'RNF8 incapaç de conjugar ubiquitines produeix uns efectes significativament atenuats en comparació amb la proteïna salvatge. Aquestes observacions suggereixen que RNF8 és un regulador negatiu del SAC i permet la reactivació de l'APC/C en condicions d'estrès mitòtic.
A més, RNF8 presenta activitat pro-apoptòtica. L'expressió ectòpica d'RNF8 en cèl·lules HeLa activa les caspases-3 i -8 i l'activitat pro-apoptòtica resultant es pot inhibir mitjançant la incubació amb els inhibidors de caspases ZVAD i ZDEVD. La mort cel·lular induïda per RNF8 es veu atenuada en gran mesura per la sobre-expressió del mutant d'RNF8 que no te activitat lligasa de ubiquitines. El tractament amb diferents agents genotòxics provoca una acumulació dosi-depenent de la proteïna RNF8 endògena que no es correlaciona amb un increment dels nivells de transcrit. Finalment, la depleció d'RNF8 permet a les cèl·lules HeLa de ser més resistents al tractament amb etopòsid, suggerint que RNF8 te un paper important regulant la mort cel·lular causada per aquesta droga.
Ubiquitylation is a post-translational protein modification, which regulates very different signaling pathways and biological processes. Ubiquitylation occurs thanks to the catalytic activity of ubiquitin activating enzymes or E1, ubiquitin conjugating enzymes or E2 and ubiquitin ligases or E3 and the substrate can be modified with one or more ubiquitin moieties that form polyubiquitin chains.
The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified two proteins, namely RNF8, KIA00675, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2's that mediate canonical (K48) polyubiquitylation. RNF8 has ubiquitin ligase autocatalytic activity that elongates chains through either K48 or K63 of ubiquitin, and UBC13 activity is required to elongate the latter chains. RNF8 and UBC13 co-localize in the nucleus.
RNF8 is a ubiquitin ligase that bears a FHA domain near its amino end, and a RING finger domain at its carboxy terminus, through which it can recruit several ubiquitin-conjugating enzymes. In metazoans, only the mitotic checkpoint regulator CHFR shares this domain architecture. RNF8 follows a cell-cycle-dependent turnover, with levels increasing from G1 to M, reaching a maximum at metaphase, followed by an abrupt decline at anaphase, and recovery of protein levels at the end of mitosis. Overexpression of RNF8 caused a reduction of the G2-M population and an accumulation of cells in G1, while siRNA depletion did not significantly alter the basal cell cycle. Depletion of RNF8 caused a delay in the exit from nocodazole-induced arrest, which was associated with a reduced turnover of the APC/C substrate cyclin B1. Conversely, cells overexpressing RNF8 failed to arrest at G2-M upon treatment with nocodazole. In addition, activation of the spindle assembly checkpoint with a brief exposure to nocodazole in combination with RNF8 overexpression prevented the localization of Mad2 to kinetochores. These functions of RNF8 were dependent on its ubiquitin ligase activity, since a ligase-inactive mutant produced significantly attenuated effects as compared to the wild-type protein. Taken together, these observations suggest that RNF8 is a negative regulator of the spindle assembly checkpoint that reactivates the APC/C under conditions of mitotic stress.
In addition,RNF8 functions as a proapoptotic protein. Ectopic expression of RNF8 in HeLa cells induced the activation of caspases 3 and 8, and the ensuing apoptosis was prevented by incubation with the caspase inhibitors ZVAD and ZDEVD. The apoptotic death induced by RNF8 was greatly attenuated by introduction of a point mutation in its RING finger domain that rendered it non-functional as a ubiquitin ligase, indicating that this function is essential for the proapoptotic activity of RNF8. Treatment with DNA-damaging agents causes a dose-dependent accumulation of endogenous RNF8 without increasing its mRNA levels. Finally, depletion of RNF8 with specific siRNA duplexes effectively prevented the apoptosis and caspase-3 up regulation induced by the topoisomerase II inhibitor etoposide, suggesting that RNF8 plays a major role in regulating apoptotic death caused by DNA damaging agent.
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Schlüter, Anne. "Funktionelle Analyse von RNF6, einer RLIM-ähnlichen Ubiquitin-Ligase in Vertebraten." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973073845.
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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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Sengupta, Sameer. "The influence of BRCA1's ubiquitin ligase activity on cell motility." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:b14af4ae-1f95-4d0c-85a8-faaf4fa950c4.
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Breast cancer type 1 susceptibility protein (BRCA1) has been established as an important tumour suppressor protein and its loss of function is associated with hereditary breast and ovarian cancer. An emerging body of work suggests that BRCA1 is involved in sporadic cases of breast and ovarian cancers and may also have a role in other cancers, indicating a more global role in tumourigenesis. BRCA1-mutated cancers can be early-onset and are characterised by being highly aggressive with a propensity to metastasize, thus from a clinical perspective there is a requirement to understand the molecular mechanisms in order to be able to tailor treatments and develop therapeutics. BRCA1 has numerous cellular functions, many ascribed to its role in maintenance of genome integrity, transcription and checkpoint control. More recently, a number of extra-nuclear roles have been established. An interesting novel function is the role of the E3 ubiquitin ligase activity on cell motility. Abrogation of the ubiquitin ligase activity of BRCA1 results in cells exhibiting a hypermotile, invasive phenotype which may help to account for the metastatic nature of BRCA1-mutated tumours. Our aim was to further elucidate BRCA1’s role in cell motility, starting with the identification of relevant candidate ubiquitin ligase substrates. To date, there has yet to be a systematic approach to identify BRCA1’s ubiquitin ligase substrates. Thus we undertook an unbiased proteomic approach to identify extra-nuclear candidates by comparing the profiles of ubiquitinated proteins in breast cancer epithelial cells expressing either functional BRCA1 or ubiquitin ligase-dead BRCA1. We identified 55 candidates which were differentially enriched between the two cell lines and through pathway analysis we determined a significant proportion were cytoskeletal and translation related proteins. Using an ubiquitin-remnant profiling approach, we also identified the site(s) of ubiquitination for many of the candidates. To assess the role of these candidates in cell motility initially we adopted an in silico approach. We used existing time-lapse movies from the online database (www.mitocheck.org) which systematically siRNA knocked down every single gene in the human genome. We developed a series of algorithms which track cell motility from these movies and used these to analyse 192,000 movies containing 3.5 billion cell steps. We have produced a complete database containing motility information after siRNA knockdown of every gene in the human genome, which has been annotated with gene ontologies, KEGG families, Gene Descriptions, SwissProt, Ensembl IDs and siRNA information. In addition to providing motility data of our candidates, we also carried out gene set enrichment analysis on the whole dataset to uncover structural or functional families that may be involved in up-regulating motility when knocked down by siRNA. This is the first report of a genome-wide motility database. Based on overlaps between the results from these two large-scale unbiased proteomic and in silico datasets, we selected 4 candidates, namely, ezrin, moesin, fermitin-2 and delta-catenin. Through monolayer wound healing, cell spreading and single cell motility assays, we determined that ezrin was a particularly relevant and informative candidate. The hypermotile phenotype observed in cells expressing ubiquitin ligase dead BRCA1 was rescued through siRNA knockdown of ezrin and thus we suggest that BRCA1 may regulate cell motility through effects on ezrin. This thesis has investigated candidate BRCA1’s role in cell motility, identified candidate substrates for the E3 ubiquitin ligase activity, established a genome-wide motility database and proposed a possible pathway through which BRCA1 may mediate cell motility and by extension metastasis.
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Megumi, Yuzuru. "Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex." Kyoto University, 2006. http://hdl.handle.net/2433/144318.
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Weber, Annika. "Functional characterization of Ubc6 and Ubc7 at the Doa10 ubiquitin ligase." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17609.
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In Saccharomyces cerevisiae nimmt die membrangebundene RING-Ub-Ligase Doa10 eine bedeutende Rolle in der Proteinqualitätskontrolle (PQC) des Endoplasmatischen Retikulums (ER) und des Nukleus ein. Doa10 katalysiert dabei die Verknüpfung K48- verbundener Ub-Ketten auf Proteine, die entweder in der ER-Membran oder löslich im Cytosol oder dem Nukleoplasma vorliegen. Diese Markierung leitet die Degradation dieser Proteine ein. Interessanterweise kooperiert Doa10, im Gegensatz zu anderen RING-Ub-Ligasen, mit zwei Ub-konjugierenden Enzymen (E2), um ihre Substrate zu prozessieren. In dieser Arbeit wird veranschaulicht, wie die beiden hochspezialisierten E2 Enzyme Ubc6 und Ubc7 sequentiell agieren, um Doa10 Substrate zu modifizieren. Zuerst wird ein einzelnes Ub-Molekül Ubc6-abhängig an ein Substrat konjugiert (Initiation). Von diesem Rest ausgehen katalysiert Ubc7 die Ausbildung einer K48-verbundenen Ub-Kette (Elongation). Die Fähigkeit von Ubc6 nicht nur Lysine, sondern auch hydroxylierten Aminosäuren wie Serin und Threonin mit Ub-Molekülen zu verknüpfen, erweitert das Substratspektrum von Doa10 und ermöglicht die Prozessieren von Proteinen, die keine zugänglichen Lysinreste exponieren. Weiterhin wird gezeigt, dass ein Überangebot von Ubc6 den Doa10-abhängigen Substratabbau beeinträchtigt. Dies weist darauf hin, dass die Generierung eines effizienten Poly-Ub-Signals einer streng kontrollierten Koordination beider E2 Enzyme am Doa10-Ligase-Komplex unterliegt.In Saccharomyces cerevisiae, the membrane-bound RING-type Ub ligase Doa10 is a key player of Protein Quality Control (PQC) in the endoplasmic reticulum (ER) and the nucleus. Doa10 promotes lysine 48-linked poly-ubiquitylation of proteins that either reside in the ER membrane or are soluble in the cytosol or the nucleus and thereby labels them for degradation. Strikingly, in contrast to other RING Ub ligases, which typically employ a single Ub conjugating enzyme (E2) for substrate ubiquitylation, the Doa10 ligase requires two of such enzymes for client processing. This study demonstrates that the highly specialized E2 enzymes Ubc6 and Ubc7 act in a sequential manner on Doa10 client proteins. In a first step Ubc6 attaches a single Ub molecule to a substrate (priming), which is followed by the elongation of this moiety with K48-linked Ub chains by Ubc7 (elongation). The ability of Ubc6 to conjugate Ub not only to lysine but also to hydroxylated amino acids like serine and threonine broadens the substrate range of Doa10 and allows processing of proteins, which do not expose accessible lysine residues. Overproduction of Ubc6 was shown to impair Doa10 dependent substrate degradation. Apparently, the generation of a productive K48-linked poly-Ub signal requires a tightly coordinated activity of the individual E2 enzymes at the Doa10 ligase complex.
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Davidge, Brittney Marie. "The Cul3 Ubiquitin Ligase: an Essential Regulator of Diverse Cellular Processes." PDXScholar, 2017. https://pdxscholar.library.pdx.edu/open_access_etds/3782.
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Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3's role in several of these pathways and provides new mechanistic details regarding the role of Cul3 in eukaryotic cells. Cyclin E is an example of a protein that is regulated in a Cul3-dependent manner. Cyclin E is a cell cycle regulator that controls the beginning of DNA replication in mammalian cells. Increased levels of cyclin E are found in some cancers, in addition, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with tumorigenesis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligase complexes, Cul1 and Cul3. Cul1 mediated degradation of cyclin E is triggered by cyclin E phosphorylation, however the mechanism Cul3 uses to ubiquitinate cyclin E is poorly understood. In order to gain a better understanding of how Cul3 mediates cyclin E destruction we identified the degron on cyclin E that is important in Cul3 dependent degradation. In addition, we show this degron is lacking in LMW cyclin E (found in abundance in breast cancer), providing a novel mechanism for how these cyclin E modifications result in increased cyclin E levels by avoiding the Cul3 degradation pathway.
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Auguste, Tiphanie. "Implication de ROQUIN dans la physiopathologie du lymphome T angio-immunoblastique." Thesis, Paris Est, 2012. http://www.theses.fr/2012PEST0076.
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Implication de ROQUIN dans la physiopathologie du lymphome T angio-immunoblastique. Le T-LAI est un lymphome T périphérique qui de part sa rareté bénéficie de peu d'études contrairement aux lymphomes B. En France, le T-LAI est le PTCL le plus fréquemment rencontré. Malgré une évolution clinique variable, le T-LAI reste une tumeur agressive dont la survie globale est inférieure à 3 ans. Un des objectifs de notre équipe est donc de mieux comprendre la physiopathologie de ce lymphome et d'identifier des évènements oncogéniques conduisant à son développement. Dans le cadre de ce projet, notre étude s'est portée sur le gène ROQUIN qui code une E3 ubiquitine ligase de la famille RING et dont la mutation est associée à l'apparition d'un syndrome proche du T-LAI chez la souris sanroque.Bien que nous n'ayons détecté aucune mutation dans la séquence codante de ROQUIN nous avons identifié un nouveau transcrit alternatif appelé ROQUIN ØE17. Celui-ci code une protéine qui, comme la forme sauvage, se localise dans les granules de stress et les corps P et interagit avec certains ARNm et micro-ARN. Néanmoins il est le seul à inhiber l'expression de la molécule de costimulation ICOS. ROQUIN ØE17, qui est présent en concentrations variables dans les différentes populations lymphocytaires T n'est quasiment pas exprimé dans les T-LAI. De ce fait, la perte du transcrit ROQUIN ØE17 pourrait participer à la genèse et/ou développement de ce lymphomeImplication of ROQUIN in the physiopathology of angio-immunoblastic T cell lymphoma. AITL is a peripheral T cell lymphoma, poorly studied compared to B cell lymphomas due to its rarity. In France, AITL is the PTCL the most frequently encountered. Despite a variable clinical course, AITL is an aggressive tumor with an overall survival lower than 3 years. One of our goal is to better understand the physiopathology of this lymphoma and identify oncogenic events that lead to its development. In this project, our study was focused on ROQUIN gene that encodes a RING E3 ubiquitin ligase and whose mutation induces an AITL-like syndrom in sanroque mice.Although we did not detect any mutation in ROQUIN coding sequence, we identified a novel alternative transcript referred as ROQUIN ØE17. It encodes a protein that, like wild type protein, localizes to stress granules and P bodies and interacts with mRNAs and microRNAs. However, only ROQUIN ØE17 inhibits the expression of the costimulatory molecule ICOS. This transcript, whose expression varies between T cell populations, is hardly expressed in AITL. Consequently, the loss of ROQUIN ØE17 could be involved in the genesis and/or development of this lymphoma
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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.
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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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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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Tucker, Wesley Owen. "Towards specific DNA aptamers which bind and inhibit WWP1 HECT ubiquitin ligase in the osteoblast." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/205640.
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DNA aptamers have been studied since their inception in 1990, but have only targeted membrane and serum proteins in therapeutics. Their potential as inhibitors of protein function is hampered by their inability to efficiently enter cells in order to function. Surmounting this hurdle is worthwhile since the inhibition of protein-protein interactions is not achievable by small molecule pharmaceuticals alone.
Herein we target an intracellular ubiquitin ligase WWP1, which is known to complex with Schnurri3 and polyubiquitinate Runx2, thus targeting it for proteosomal destruction. Since Runx2 is the key transcriptional regulator of osteoblast differentiation, WWP1 inhibition may encourage osteoblast differentiation, and by extension force bone deposition in osteoporosis sufferers. By targeting WWP1 we attempt to intervene in intracellular protein interactions with an aptamer for the first time.
To begin this effort we cloned, expressed, and purified three functionally important truncations of WWP1. The final protein pools were highly concentrated above 2 mg/mL, approximately 95% pure, and were found to be acceptably soluble after assessment in various buffers. DNA aptamers were then selected against these WWP1 truncations using the established SELEX method while monitoring the progression of the enrichment with PCR. After 12 selection rounds of increasing stringency, pools were sequenced and assessed for homogeneity and secondary structure. Several groups of enriched and identical DNA sequences were obtained with no obvious pattern in secondary structure seen between them. While focusing on sequences specific for the active site containing C-lobe, we then evaluated the aptamers for their ability to bind key functional regions of WWP1 and inhibit its function as an enzyme. For the most potent aptamer from the C-lobe pool, an Electrophoretic Mobility Shift Assay (EMSA) estimated a Ki of around 2 μM. Furthermore, a HECT ubiquitin ligase activity assay was developed to evaluate inhibition, and an IC50 of around 100 μM was found for the most inhibitory of three C-lobe aptamers. This aptamer was then transfected into SaOS-2 osteoblastic cells so that localization could be assessed with fluorescence microscopy. Surprisingly, both the C-lobe specific aptamer and a control sequence were found to enter the cells with or without the employment of transfection reagent. Moreover, approximately 60% migrated to the nucleus and remained there over a period of days, which implies diffusion through the Nuclear Pore Complex. Taken together, this work introduces an alternative approach to disease therapy by targeting intracellular proteins with aptamers, and may have significant implications for expanding the therapeutic applications of nucleic acid aptamers in the future.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
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Plant, Pamela J. "A role for the C2 domain of the ubiquitin-protein ligase Nedd4." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0026/NQ49821.pdf.
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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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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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Arimoto, Keiichiro. "Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125." Kyoto University, 2009. http://hdl.handle.net/2433/124282.
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Snyder, Anita Kay. "Characterization of the gene Arabidopsis Ankyrin RING 3 cloning and ubiquitin ligase activity /." [Gainesville, Fla.] : University of Florida, 2001. http://purl.fcla.edu/fcla/etd/UFE0000354.
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Thesis (M.S.)--University of Florida, 2001.Title from title page of source document. Document formatted into pages; contains viii, 66 p.; also contains graphics. Includes vita. Includes bibliographical references.
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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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Jandke, Anett. "The role of the c-Jun ubiquitin ligase Fbw7 in the nervous system." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444455/.
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Fbw7 belongs to the family of F-box proteins, which function as substrate recognition subunits of SCF complexes. Fbw7 controls the stability of several proteins including cyclin E, the Notch intracellular domain and c-Myc. In 2004 our lab additionally identified phospho-c-Jun as an Fbw7 substrate, c-Jun is part of the AP-1 transcription complex, whose activity is strongly induced in response to numerous signals such as growth factors, cytokines and extracellular stresses. Furthermore elevated phospho-c-Jun levels induce neuronal apoptosis. To investigate the significance of c-Jun regulation by Fbw7 in the nervous system, I generated mice harbouring a floxed fbw7 allele, fbw7. fbw7 mice were bred to various Cre transgenic lines that express the Cre recombinase under nervous system specific promoters to obtain mice with a tissue specific deletion of Fbw7. I confirmed published results that ubiquitous deletion of Fbw7 mediated by PGK-Cre is lethal. To delete Fbw7 at the stage of neuronal precursors, fbwff mice were crossed to the Nestin-cre line (fbw7). These mice die perinatally and show an increase in apoptosis at El6. As the lethality of the fbw7AN mice does not allow the investigation of Fbw7 in the adult nervous system, further crosses, using other cre-transgenic lines, were set up. Fbw7 deletion in postmitotic neurons (fbw7ApN) causes a Parkinson's disease like phenotype with a severe hindlimb tremor and a reduced cortical cellularity. Fbw7 deletion in the cerebellar vermis (fbw7ACb) resulted in cerebella that are characterised by a reduced size, foliation defects accompanied by an astrocytic gliosis and a phospho-c-Jun dependent Purkinje cell loss. Concomitant deletion of c-Jun in the cerebellum (fbw7ACb :c-junACb) partially rescues the cerebellar phenotype caused by Fbw7 deletion. Thus the data in this thesis demonstrate a role for Fbw7 in cerebellar development and the central nervous system and identify c-Jun as an essential Fbw7 substrate in the nervous system.
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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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Sabri, Tarek. "Interaction of the MYST family of lysine acetyltransferases with the RNF8 ubiquitin ligase." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121507.
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Lysine acetyltransferases (KATs) are post-translational modifiers that catalyze acetylation of histones and non-histone proteins at specific lysine residues. The MYST family in humans consists of five proteins MOZ, MORF, Tip60, MOF, and HBO1. MYST proteins have diverse biological activities, ranging from regulation of cell homeostasis to chromatin structure across broad chromosomal domains. Ubiquitination is another posttranslational modification that has similar biological features as acetylation. However, the process of ubiquitination requires three enzymes to transfer the ubiquitin tag to target proteins: an E1 (ubiquitin activating enzyme), an E2 (conjugating enzyme), and an E3 ligase that determines the specificity of ubiquitination. E3 ligases are involved in many cellular processes similar to MYST proteins. Recently, the E3 ligase RNF8 has been identified to play a significant role in DNA damage repair. So far, only a limited number of substrates and cellular functions have been assigned to RNF8. In searching for additional substrates, we have found that all MYST family are interaction partners for RNF8. To gain a better functional understanding of this binding we tested if RNF8 could modulate acetylation activity. Indeed, our data revealed that RNF8 binding has the ability to regulate acetylation activity of all MYST members. Due to the importance of the MYST family and RNF8 in regulating many cellular functions, our findings shed light on new regulatory mechanisms of histone modifiers that carry out epigenetic modifications responsible in shaping many cellular activities.Les lysine acétyltransferases (KATs) sont des modificateurs post-traductionneles qui catalysent l'acétylation sur des résidus de lysine spécifiques dans des protéines histones et non-histones. Chez l'humain, la famille MYST se compose de cinq protéines que sont MOZ, MORF, Tip60, MOF, et HBO1. Ces protéines MYST ont des activités biologiques diverses, allant de la régulation de l'homéostasie cellulaire à celle de la structure de la chromatine dans des domaines chromosomiques. L'ubiquitination est une autre modification post-traductionnelle qui a des caractéristiques biologiques similaires à celles de l'acétylation, mais dont le processus requière trois enzymes pour le transfert de l'étiquette ubiquitine sur les protéines cibles. Ces enzymes sont E1 (enzyme activatrice d'ubiquitine), E2 (enzyme de conjugaison), et E3, une ligase qui détermine la spécificité de l'ubiquitination. Les ligases E3 sont impliquées dans de nombreux processus cellulaires semblables à ceux des protéines MYST. Récemment, la ligase E3 RNF8 a été identifiée comme jouant un rôle important dans la réparation de l'ADN. Jusqu'à présent, seul un nombre limité de substrats ont été attribués à la protéine RNF8. En cherchant de nouveaux substrats, nous avons observé que toutes les protéines de la famille MYST sont des partenaires d'interaction pour RNF8. Afin d'avoir une meilleure compréhension de la fonction de ces interactions, nous avons fait des tests pour vérifier si RNF8 peut moduler l'activité d'acétylation. Nos données ont démontré que l'interaction avec RNF8 a la capacité de réguler l'activité d'acétylation de toutes les protéines de la famille MYST. Considérant l'importance des protéines MYST et de RNF8 dans la régulation de nombreuses fonctions cellulaires, nos résultats mettent en lumière de nouveaux mécanismes de régulation des modificateurs d'histones. Soulignons que ceux-ci effectuent des modifications épigénétiques responsables de l'élaboration de nombreuses activités cellulaires.
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Yim, Hiu. "Investigating the role of WWP2 ubiquitin ligase isoforms in TGFβ-dependent oncogenic signalling." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/67062/.
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Ubiquitination is a post-translational modification involving the attachment of ubiquitin molecules onto target substrates. The most common function of ubiquitination is proteasomal degradation, which is commonly facilitated by the ubiquitination of target proteins at Lys-48. E3 ligases are crucial to the process of ubiquitination, and include WWP2 ubiquitin Ligase, an E3 enzyme that has been shown to interact with Smad mediators of the TGF family and might subsequently have a role in TGFβ-dependent oncogenic processes such as EMT. To elucidate the function of WWP2 in TGFβ-signalling and increase our understanding of this protein, is therefore the forefront of our research. Here, we found that the overexpression of WWP2 WW3-4 recognition domain led to an increase in Smad3-dependent TGF gene expression, providing strong evidence that in the absence of TGF , WW3-4 binds to the Smad3 mediator. We also predicted and validated the expression of two novel WWP2 isoforms, WWP2-ΔHECT and WWP2-N-ΔC2. Results suggest that the incomplete HECT domain within WWP2-ΔHECT is catalytically non-functional. Furthermore, the transcription of WWP2-ΔHECT has been linked to a 2kb region within intron 9/10 with its expression positively regulated by EGF stimulation. On the contrary, when the transcriptional mechanism of the existing WWP2-C isoform was investigated, a 0.5kb region at the end of intron 10/11 was identified to enhance promoter activity responsible for expression, which was negatively regulated by the SOX9 transcription factor. It was also found that ESRP splicing factor is involved in the negative regulation of not only the existing isoform WWP2-N, but also the novel WWP2-ΔHECT isoform. Using expression studies, we found that WWP2-N and ΔHECT were expressed at higher levels in epithelial cells, therefore suggesting their role as guardians of the epithelial phenotype during EMT. Overall, the data provided here can help clarify the role of WWP2 in TGFβ-dependent oncogenic signalling.
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Klejnot, Marta. "Structural and biochemical insights into members of the kinesin and ubiquitin ligase families." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/5108/.
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The thesis focuses on characterisation of two different protein families, kinesins and ubiquitin ligases, that are involved in different biological processes. Kinesins constitute a superfamily of microtubule-based motor proteins, fulfilling important cellular functions, ranging from intracellular transport to cell division. They also play a role in primary cilia and Hedgehog signalling. Ubiquitin ligases are enzymes that catalyse the transfer of ubiquitin to the lysine residues of the substrate. Marking protein substrates with ubiquitin alters their functions and fates. With nearly 700 different ubiquitin ligases in humans, they control a vast array of cellular processes. The first part of this thesis summarises results relating to kinesins. Protocols for creation of a panel of kinesin motor domains, a useful tool for anti-mitotic inhibitors specificity testing are presented. Furthermore, biochemical, kinetic and structural features of a poorly described kinesin motor protein - Kif15 are reported. Kif15's motor domain structure is described and compared to Eg5's catalytic core. Moreover, the influence of Eg5 inhibitors on Kif15 ATPase activity is investigated. Scouting for small molecules targeting Kif15 is also performed. Kif15 interaction with microtubules in various nucleotide-bound states is characterised. The possibility of a secondary microtubule binding site in the tail of Kif15 is examined. The binding of Kif15 with partner proteins is also investigated. Additionally the high-resolution structure of the human Kif7 motor domain is presented, providing the first step towards structural characterisation of this Kinesin-4 family member. The second part of this thesis concerns a ubiquitin ligase, Trim28. Trim28 was first reported as a transcription corepressor, working by recruiting proteins that drive the heterochromatin state, whilst its mechanism of action as a ubiquitin ligase remains elusive. Attempts to crystallise and determine the three-dimensional structure of Trim28 are described. Additionally, Trim28 functioning as an E3 ubiquitin ligase, and its interactions with E2s, KRAB domains of various zinc finger proteins and with the Mage-C2 protein are investigated. The results provide the foundation for future studies on Trim28.