The ubiquitination of proteins serves as molecular signal to control an enormous number of physiological processes and its dysregulation is connected to human diseases like cancer. The versatility of this signal stems from the diverse ways by which ubiquitin can be attached to its targets. Thus, specificity and tight regulation of the ubiquitination are pivotal requirements of ubiquitin signaling. Ubiquitin-conjugating enzymes (E2s) act at the heart of the ubiquitination cascade, transferring ubiquitin from a ubiquitin-activating enzyme (E1) to a ubiquitin ligase (E3) or substrate. When cooperating with a RING-type E3, ubiquitin-conjugating enzymes can determine linkage specificity in ubiquitin chain formation. Our understanding of the regulation of E2 activities is still limited at a structural level. The work described here identifies two regulation mechanisms in UBE2S, a cognate E2 of the human RING-type E3 anaphase-promoting complex/cyclosome (APC/C). UBE2S elongates ubiquitin chains on APC/C substrates in a Lys11 linkage-specific manner, thereby targeting these substrates for degradation and driving mitotic progression. In addition, UBE2S was found to have a role in DNA repair by enhancing non-homologous end-joining (NHEJ) and causing transcriptional arrest at DNA damage sites in homologous recombination (HR). Furthermore, UBE2S overexpression is a characteristic feature of many cancer types and is connected to poor prognosis and diminished response to therapy. The first regulatory mechanism uncovered in this thesis involves the intramolecular auto-ubiquitination of a particular lysine residue (Lys+5) close to the active site cysteine, presumably through conformational flexibility of the active site region. The Lys+5-linked ubiquitin molecule adopts a donor-like, ‘closed’ orientation towards UBE2S, thereby conferring auto-inhibition. Notably, Lys+5 is a major physiological ubiquitination site in ~25% of the human E2 enzymes, thus providing regulatory opportunities beyond UBE2S. Besides the active, monomeric state and the auto-inhibited state caused by auto-ubiquitination, I discovered that UBE2S can adopt a dimeric state. The latter also provides an auto-inhibited state, in which ubiquitin transfer is blocked via the obstruction of donor binding. UBE2S dimerization is promoted by its unique C-terminal extension, suppresses auto-ubiquitination and thereby the proteasomal degradation of UBE2S. Taken together, the data provided in this thesis illustrate the intricate ways by which UBE2S activity is fine-tuned and the notion that structurally diverse mechanisms have evolved to restrict the first step in the catalytic cycle of E2 enzymes. / Die Ubiquitinierung von Proteinen fungiert als molekulares Signal zur Kontrolle einer Vielzahl physiologischer Prozesse, wobei eine gestörte Regulation der Ubiquitinierung eng mit zahlreichen Erkrankungen, wie beispielsweise Krebs, verbunden ist. Aufgrund der verschiedenen Verknüpfungsmöglichkeiten von Ubiquitin, die das zelluläre Schicksal des Zielproteins bestimmen, sind Spezifität und stringente Regulation unabkömmliche Voraussetzungen im Ubiquitinierungsprozess. Ubiquitin-konjugierende Enzyme (E2s) fungieren in der Mitte der Ubiquitinierungskaskade. Sie übernehmen ein Ubiquitinmolekül vom Ubiquitin-aktivierenden Enzym (E1) und übertragen es auf eine Ubiquitin-Ligase (E3) oder direkt auf das Zielprotein. Arbeiten Ubiquitin-konjugierende Enzyme mit E3s des RING-Typus zusammen, so bestimmen E2s die Art der Verknüpfung. Die Regulation der Aktivität Ubiquitin-konjugierender Enzyme auf struktureller Ebene ist jedoch bisher nur bedingt verstanden. Die hier dargelegte Arbeit umfasst die Identifizierung zweier Regulationsmechanismen des Ubiquitin-konjugierenden Enzyms UBE2S. UBE2S arbeitet mit einem humanen E3 des RING-Typus‚ dem ‚Anaphase Promoting Complex/Cyclosome‘ (APC/C) zusammen und bildet Lys11-spezifische Ubiquitinketten auf Substraten des APC/Cs. Hierdurch werden die Substrate für den Abbau durch das Proteasom markiert, was das Fortschreiten der Mitose bedingt. Zusätzlich wird UBE2S eine Rolle in der DNS-Reparatur zugeschrieben. Hierbei verstärkt UBE2S die nicht-homologe Rekombination (NHEJ) und verhindert außerdem die Transkription an DNS-Bruchstellen, die durch Homologe Rekombination (HR) repariert werden. Die Überexpression von UBE2S ist ein Charakteristikum verschiedenster Krebsarten, vermindert den Erfolg herkömmlicher Krebstherapien, und führt somit zu schlechten Prognosen für betroffenen Patienten. Der erste hier beschriebene Regulationsmechanismus beinhaltet die intramolekulare Ubiquitinierung eines Lysins (Lys+5) nahe des katalytischen Cysteins, mutmaßlich durch strukturelle Flexibilität der Region des aktiven Zentrums. Das Lys+5-verknüpfte Ubiquitin nimmt eine Donorubiquitin-ähnliche Position auf UBE2S ein, wodurch UBE2S gehemmt wird. Da ein Lysin an der Position +5 in ~25% der humanen E2-Enzyme vorhanden und eine physiologische Ubiquitinierungsstelle ist, birgt dieser Mechanismus Regulationsmöglichkeiten über UBE2S hinaus. Zusätzlich zum aktiven monomeren Zustand und dem durch Autoubiquitinierung ausgelösten inhibierten Zustand, kann UBE2S auch als Dimer vorliegen. In diesem Zustand ist es ebenfalls inaktiv, da die Donorubiquitin-Bindestelle auf UBE2S durch ein zweites Molekül des E2s blockiert wird. Begünstigt wird die Dimerisierung durch die C-terminale Verlängerung von UBE2S und verhindert so deren Autoubiquitinierung, und folglich den proteasomalen Abbau von UBE2S. Es handelt sich hierbei somit um einen zweiten Regulationsmechanismus von UBE2S. Zusammenfassend veranschaulichen die in dieser Arbeit dargelegten Daten die komplexen Möglichkeiten, durch die die Aktivität von UBE2S reguliert werden kann, sowie die Erkenntnis, dass strukturell unterschiedliche Mechanismen existieren, um den ersten Schritt der von Ubiquitin-konjugierenden Enzymen katalysierten Reaktion zu hemmen.

The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...

Solid-state NMR serves as a powerful method for investigating atomic-level details of insoluble biomolecules, enabling the determination of protein 3D structures and probing molecular motions across a broad range of timescales. In this thesis, I present structural studies on a novel heterotypic and functional amyloid, dynamics studies, and chemical shift anisotropy studies of a microcrystalline protein, ubiquitin. In Chapter 1, I provide a summary of the main interactions in solid-state NMR and discuss relevant pulse sequences employed in this thesis. Chapter 2 briefly explores the characteristic properties of amyloids, highlighting well-studied examples of disease-related and functional amyloids. Special treatments employed in amyloid structure determination using solid-state NMR are also summarized. Chapter 3 presents structural studies on a heterotypic functional amyloid, mcmvM45-hsRIPK3, where M45 is a protein encoded by murine cytomegalovirus (MCMV) and RIPK3 is from humans. Both M45 and RIPK3 belong to a family of RHIM-containing proteins, which are involved in innate immunity and immune response through necroptosis. SSNMR data on various isotopically labeled samples enable the chemical shift assignment for both M45 and RIPK3, providing intra- and inter-molecular contacts. By combining these constraints, we calculate the structure of the hetero-amyloid M45-RIPK3, reporting two structures distinct from RIPK1-RIPK3. In Chapter 4, I measure backbone 15N-13CO order parameters of microcrystalline ubiquitin using DCP-REDOR. Two isotopically labeled samples, 1-13C-glucose and 1,3-13C-glycerol, D₂O labeled, are studied and compared, identifying mobile residues and assessing the effect of isotropic labeling on the measurements of backbone 15N-13Co order parameters. Experimental order parameters are compared with a 1μs MD simulation for insights. Chapter 5 focuses on the chemical shift anisotropy (CSA) of uniformly labeled microcrystalline ubiquitin using a novel pulse sequence allowing the measurement of large CSAs under practical conditions. We explore CSA parameter trends, correlations between isotropic shifts, and hydrogen bond geometries. Comparison with solution-NMR results demonstrates high consistencies with asymmetry parameters (η), providing insights into the motion modes of microcrystalline proteins alongside order parameter measurements. Chapter 6 provides a comprehensive summary of the conclusions drawn from the preceding chapters, while also outlining future directions for each project.

Obesity is a major health condition in which little is known about the molecular mechanisms that drive it. The hypothalamus is the primary control center for controlling both food intake and energy expenditure in order to maintain the body's energy balance and dysregulation of molecular processes in this region have been implicated in the development and progression of obesity. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. Here, we used a rodent diet-induced obesity model in combination with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic 1) DNA 5-hmC levels, 2) Pomc methylation, and 3) dysregulated ubiquitin-proteasome signaling in abnormal weight gain following exposure to obesogenic diets. We found that males, but not females, have decreased levels of DNA 5-hmC in the hypothalamus following exposure to a high fat diet, which tracked body weight. Short-term exposure to a high fat diet, which does not result in significant weight gain, resulted in decreased hypothalamic DNA 5-hmC levels, suggesting these changes occur prior to obesity development. Moreover, decreases in DNA 5-hmC persist even after the high fat diet is removed. Importantly, CRISPR-dCas9 mediated upregulation of DNA 5-hmC enzymes in the male, but not female, hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. Next, we used the CRISPR-dCas9-TET1 and dCas9-DNMT3a systems to test the role of Pomc DNA methylation in the hypothalamus in abnormal weight gain following acute exposure to a high fat diet in male rats. We found that exposure to a high fat diet increases Pomc DNA methylation and reduces gene expression in the hypothalamus. Despite this, we found that CRISPR-dCas9-TET1-mediated demethylation of Pomc was not sufficient to prevent abnormal weight gain following exposure to a high fat diet. Moreover, CRISPR-dCas9-DNMT3a-mediated methylation of Pomc did not alter weight gain following exposure to standard or high fat diets. Finally, we found that both males and females showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. However, while the CRISPR-dCas9 system was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. Collectively, this data reveals novel, sex-specific differences in the engagement of the ubiquitin proteasome system and role of DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype. / Doctor of Philosophy / Obesity affects 34% of the American population at an annual cost of more than $340 billion in healthcare and is a risk factor for the development of diabetes and certain cancers. Genetic and environmental factors have also been shown to influence the expression of genes that play a role in the development of obesity. The hypothalamus coordinates many integral activities such as hormone regulation and feed intake and numerous studies have observed altered hypothalamic gene regulation in obesity models. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. In this document, I outline a series of experiments designed to elucidate novel, sex-specific differences in the role of the ubiquitin proteasome system and DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype.

Yes / The realisation that unregulated activation of the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway is a key driver of a wide range of diseases has identified its components as targets for therapeutic intervention by small molecule inhibitors and biologicals. In this review, we discuss JAK-STAT signalling pathway inhibition by the inducible inhibitor “suppressor of cytokine signaling 3 (SOCS3), its role in diseases such as myeloproliferative disorders, and its function as part of a multi-subunit E3 ubiquitin ligase complex. In addition, we highlight potential applications of these insights into SOCS3-based therapeutic strategies for management of conditions such as vascular re-stenosis associated with acute vascular injury, where there is strong evidence that multiple processes involved in disease progression could be attenuated by localized potentiation of SOCS3 expression levels. / British Heart Foundation; Chief Scientist's Office; NHS Greater Glasgow and Clyde Research Endowment Fund; BBSRC

The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors. / Der Krankheitserreger Chlamydia trachomatis (CT) - ein gramnegatives Bakterium - ist verantwortlich für die häufigste sexuell übertragene Infektionskrankheit weltweit, die CT basierte Chlamydiose. Sie wird von der Weltgesundheitsorganisation zu den vernachlässigten Krankheiten gezählt. CT Infektionen können unter anderem zu Unfruchtbarkeit, Erblindung und reaktiver Arthritis führen. CT agiert als Krankheitserreger mittels seiner Fähigkeit, die Immunantwort des Wirts zu umgehen. Dies umfasst unter anderem die Schwächung und Störung der NF-κB vermittelten Entzündungsantwort und des Mcl1 pro-Apoptoseweges über ihr deubiquitinierendes, deneddylierendes und trans-acetylierendes Enzym ChlaDub1 (Cdu1). Die Expression von Cdu1 ist aber auch mit der Fragmentierung des Golgi-Apparates des Wirtes verknüpft, ein Schlüsselprozess bei Infektionen mit CT. Cdu1 ist daher vermutlich ein attraktives Zielprotein für die Entwicklung von Wirkstoffen, um CT Infektionen zu behandeln. Eine Leitstrukturverbindung zur Entwicklung neuer wirksamer Inhibitoren war bislang jedoch noch nicht bekannt. Sequenzvergleiche und phylogenetische Untersuchungen verorten Cdu1 im CE Clan der Cysteinproteasen. Die Adenovirus-Protease (Adenain) gehört ebenfalls diesem Clan an und besitzt strukturelle Ähnlichkeit mit Cdu1. Unter Ausnutzung der topologischen Ähnlichkeiten der aktiven Zentren von Cdu1 und Adenain wurde ein Target-Hopping Ansatz mit einem klar definierten und fokussierten Satz von bei Novartis entwickelten Adenain-Inhibitoren verfolgt. Die hierbei identifizierten Cyano-Pyrimidine stellen die ersten kovalenten Inhibitoren von Cdu1 dar, die an das aktive Zentrum von Cdu1 binden und es direkt adressieren. Hochauflösend wurden Kristallstrukturen sowohl von Komplexen von Cdu1 mit kovalent gebundenen Cyano-Pyrimidinen als auch mit Cdu1’s natürlichem Substrat Ubiquitin bestimmt. Die Kristallstrukturdaten dieser Doktorarbeit in Kombination mit Enzymassays und kovalenten Docking-Studien liefern wertvolle Hinweise bezüglich der Aktivität des Enzyms, der molekularen Substraterkennung, der Flexibiliät der Proteintasche rund um das aktive Zentrum und potentielle Hotspots für die Wechselwirkung mit Liganden. Ein strukturbasiertes Wirkstoffdesign erlaubte die Optimierung des Cyano-Pyrimidin-basierten Molekülgerüstes, die zu der Entwicklung der HJR108 Verbindung führte. Es ist das erste Molekül seiner Art, das speziell dazu entworfen wurde Cdu1 zu inhibieren. Strukturen potentiell noch wirksamerer und selektiver Cdu1 Inhibitoren werden in dieser Arbeit vorgeschlagen. Diese Dissertationsschrift liefert somit wertvolle Beiträge zum Verständnis der strukturellen Grundlagen der molekularen Erkennung von Ubiquitin durch Cdu1 und Hinweise, die die Entwicklung hoch-spezifischer kovalenter Cdu1 Inhibitoren erlauben sollten.

Teng, Man Kuen. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-169). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Declaration --- p.ii / Acknowledgments --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / List of Abbreviation --- p.viii / Table of Contents --- p.x / List of Figures --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Biochemistry of the Renin-Angiotensin System --- p.2 / Chapter 1.2 --- Physiological Roles of Angiotensin II --- p.7 / Chapter 1.3 --- Physiological Roles of Angiotensin Receptors --- p.9 / Chapter 1.4 --- Characterization of Type 2 Angiotensin Receptor --- p.12 / Chapter 1.5 --- Trafficking of Type 2 Angiotensin Receptor --- p.16 / Chapter 1.6 --- SUMO and protein SUMOylation --- p.19 / Chapter 1.7 --- Aims of Study --- p.21 / Chapter Chapter 2 --- Preparation of EGFP-and FLAG tagged wild-type AT2 and K343R-AT2 mutant constructs / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials --- p.27 / Chapter 2.2.1 --- Chemicals --- p.27 / Chapter 2.2.2 --- Enzymes --- p.27 / Chapter 2.2.3 --- DNA Purification Kit --- p.27 / Chapter 2.3 --- Methods --- p.28 / Chapter 2.3.1 --- Preparation of pEGFP/AT2 Construct --- p.28 / Chapter 2.3.1.1 --- PCR amplification --- p.30 / Chapter 2.3.1.2 --- Agarose gel electrophoresis --- p.30 / Chapter 2.3.1.3 --- Restriction enzyme digestion --- p.31 / Chapter 2.3.1.4 --- Purification of DNA fragment by ethanol precipitation --- p.31 / Chapter 2.3.1.5 --- Ligation --- p.32 / Chapter 2.3.1.6 --- Preparation of competent cells --- p.33 / Chapter 2.3.1.7 --- Bacterial transformation --- p.33 / Chapter 2.3.1.8 --- Minipreparation of　plasmid DNA --- p.34 / Chapter 2.3.1.9 --- Quantitation of DNA --- p.35 / Chapter 2.3.1.10 --- DNA sequencing --- p.36 / Chapter 2.3.2 --- Preparation of pEGFP/AT2-01igo Construct --- p.36 / Chapter 2.3.2.1 --- PCR amplification of AT2-oligo --- p.39 / Chapter 2.3.2.2 --- PCR amplification of oligo-GFP --- p.39 / Chapter 2.3.2.3 --- Overlapping PCR amplification --- p.40 / Chapter 2.3.2.4 --- Gel extraction of DNA fragment --- p.41 / Chapter 2.3.2.5 --- Restriction enzyme digestion --- p.41 / Chapter 2.3.2.6 --- Ligation and transformation --- p.42 / Chapter 2.3.2.7 --- Construction of pEGFP/oligo --- p.42 / Chapter 2.3.3 --- Preparation of pCMV/AT2 Construct --- p.43 / Chapter 2.3.3.1 --- PCR amplification --- p.45 / Chapter 2.3.3.2 --- Restriction enzyme digestion --- p.45 / Chapter 2.3.3.3 --- Ligation and transformation --- p.45 / Chapter 2.3.4 --- Preparation of mutants --- p.46 / Chapter 2.3.4.1 --- Site directed mutagenesis at SUMOylation site --- p.46 / Chapter 2.3.4.2 --- Transformation of mutants --- p.47 / Chapter 2.4 --- Results --- p.48 / Chapter 2.4.1 --- Preparation of pEGFP/AT2 Construct --- p.48 / Chapter 2.4.2 --- Preparation of pEGFP/AT2-oligo Construct --- p.50 / Chapter 2.4.3 --- Preparation of pCMV/AT2 Construct --- p.53 / Chapter 2.4.4 --- Preparation of Mutants --- p.55 / Chapter 2.5 --- Discussion --- p.57 / Chapter Chapter 3 --- Transient Expression of AT2 and K343R mutants in CHO-K1 and HEK-293 cells / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials --- p.64 / Chapter 3.2.1 --- Chemicals --- p.64 / Chapter 3.2.2 --- Antibodies --- p.64 / Chapter 3.2.3 --- Protein Concentration Measurement Kit --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Expression of AT2 in Mammalian Cells --- p.66 / Chapter 3.3.1.1 --- Cell culture --- p.66 / Chapter 3.3.1.2 --- Counting cells --- p.67 / Chapter 3.3.1.3 --- Transient transfection --- p.67 / Chapter 3.3.2 --- Western Blot Analysis --- p.68 / Chapter 3.3.2.1 --- Preparation of protein sample from total lysate --- p.68 / Chapter 3.3.2.2 --- Protein sample derived from immunoprecipitation --- p.69 / Chapter 3.3.2.3 --- SDS PAGE and Western blot analysis --- p.70 / Chapter 3.3.3 --- Confocal microscopy --- p.71 / Chapter 3.4 --- Results --- p.73 / Chapter 3.4.1 --- Expression Analysis of GFP-tagged AT2 --- p.73 / Chapter 3.4.1.1 --- Western blot analysis with anti-GFP antibody --- p.73 / Chapter 3.4.1.2 --- Western blot analysis with anti-AT2 antibody --- p.79 / Chapter 3.4.1.3 --- Confocal microscopy --- p.81 / Chapter 3.4.2 --- Western Blot Analysis of FLAG-tagged AT2 --- p.90 / Chapter 3.5 --- Discussion --- p.92 / Chapter Chapter 4 --- Stable Expression of AT2 and K343R mutants in CHO-K1 cells / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials --- p.99 / Chapter 4.2.1 --- Chemicals --- p.99 / Chapter 4.2.2 --- Enzymes --- p.99 / Chapter 4.2.3 --- Antibodies --- p.99 / Chapter 4.2.4 --- Protein Concentration Measurement Kit --- p.100 / Chapter 4.3 --- Methods --- p.101 / Chapter 4.3.1 --- Linearization of Vector --- p.101 / Chapter 4.3.2 --- Transfection by Lipofectamine 2000 --- p.101 / Chapter 4.3.3 --- Screening for the Stably Transfected Cells --- p.101 / Chapter 4.3.4 --- Western Blot Analysis --- p.103 / Chapter 4.3.5 --- Confocal Microscopy --- p.103 / Chapter 4.4 --- Results --- p.104 / Chapter 4.4.1 --- Stable expression of wild type and mutant AT2-GFP in CHO-K1 --- p.104 / Chapter 4.4.2 --- Stable expression of wild type and mutant AT2-Gly10Ser5-GFP in CHO-K1 --- p.115 / Chapter 4.4.3 --- Stable expression of wild type and mutant AT2-FL AG in CHO-K1 --- p.123 / Chapter 4.5 --- Discussion --- p.125 / Chapter Chapter 5 --- Co-immunoprecipitation Analysis of CHO-K1 stably expressing wild type and mutant AT2-Gly10Ser5-GFP / Chapter 5.1 --- Introduction --- p.129 / Chapter 5.2 --- Materials --- p.129 / Chapter 5.2.1 --- Chemicals --- p.130 / Chapter 5.2.2 --- Antibodies --- p.130 / Chapter 5.2.3 --- Protein Concentration Measurement Kit --- p.130 / Chapter 5.3 --- Methods --- p.131 / Chapter 5.3.1 --- Transfection by Lipofectaime 2000 --- p.131 / Chapter 5.3.2 --- Western Blot Analysis --- p.131 / Chapter 5.4 --- Results --- p.132 / Chapter 5.4.1 --- Western blot analysis of SUMO 1 transfected stable cell lines --- p.132 / Chapter 5.4.2 --- Western blot analysis of SUM03 transfected stable cell lines --- p.136 / Chapter 5.5 --- Discussion --- p.143 / Chapter Chapter 6 --- General Discussion / Chapter 6.1 --- Investigation of AT2 trafficking in mammalian cells --- p.147 / Chapter 6.2 --- Future Aspects --- p.153 / Appendix I Buffer composition --- p.155 / Appendix II Sequence of Primers --- p.156 / Appendix III Sequencing Results --- p.157 / References --- p.160

Polyglutamine (polyQ) diseases describe a group of late-onset progressive neurodegenerative disorders which are caused by the CAG triplet repeat expansion in the coding region of disease genes. Such expansions result in expanded polyQ tracts in the disease proteins which confer neurotoxicity. To date, nine such diseases are reported including Huntington's disease and several types of spinocerebellar ataxias. Misfolding of polyQ proteins and formation of intracellular SDS-insoluble protein aggregates are closely associated with the toxicity of these diseases. In particular, impairment of the ubiquitin-proteasome system (UPS) which is responsible for protein degradation has been observed in polyQ diseases. Recently, ubiquitin ligases, which govern substrate specificity of the UPS, have gained huge attention in polyQ disease pathogenesis studies. In humans, cullin (Cul) proteins, including Cul1, 2, 3, 4 & 5, are integral components of a group of ubiquitin ligases called cullin- RING ubiquitin ligases (CRLs). Each CRL displays distinct substrate specificity through specific substrate receptors. Cullin proteins are evolutionarily conserved and Cul orthologues are found in the Drosophila genome. In the present study, it was found that individual Culs displayed distinct effects on polyQ pathogenesis in Drosophila polyQ models. Particularly, it was found that Cul1 modulated polyQ-induced toxic phenotype. This modification was accompanied with an alteration in the ubiquitination level and SDS-solubility properties of expanded polyQ protein. Through genetic interaction studies and biochemical analyses, it is suggested that Cul1-based CRL specifically targets SDS-insoluble species of expanded polyQ protein for ubiquitination via selective recognition by CG2010 substrate receptor. On the other hand, it was found that expanded polyQ protein induced accumulation of CRL substrates in cells. Current data support a hypothesis that polyQ protein would impair the ubiquitin ligase activity of CRLs upon expansion of the polyQ domain, through interfering with neddylation of cullin and other uncharacterized mechanisms. Taken together, the present study identifies Cul1-CRL as a novel E3 ligase that modifies polyQ toxicity through modulating ubiquitination of expanded polyQ protein, and demonstrates a pathological mechanism by expanded polyQ protein through impairing CRL activity. These findings would lead to a better understanding of polyQ pathogenesis and give insights on developing treatments against polyQ diseases. / Wong, Kam Yan. / Adviser: Ho-Yin Edwin Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 260-273). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR

Sna3p and Sna4p are small proteins of unknown function possessing two transmembrane domains and belong to a small family of conserved proteins present in plant and fungi. The budding yeast has four SNA proteins (Sna1–4) that have different localizations in the cell. Sna3p is targeted to the vacuolar lumen by the multivesicular body pathway. Two observations marked Sna3p as a multivesicular body cargo that is sorted in an ubiquitin-independent manner. First, Sna3p-GFP is still correctly transported to internal multivesicular body vesicles under conditions of ubiquitin depletion, which impairs multivesicular body sorting of certain other cargoes. Second, a mutant form of Sna3p-GFP lacking the only potential positions for ubiquitylation is still correctly targeted to the vacuolar lumen. It has thus been postulated that ubiquitylation marks, but not all, membrane proteins for sorting into the interior of the vacuole. In this study we present a further characterization of the Golgi to vacuole trafficking of Sna3p together with its ubiquitylation status. We observed that Sna3p physically interacts with the E3 ligase Rsp5p and that this interaction is essential for sorting of Sna3p to the endosomal pathway. Sna3p is ubiquitylated on its Lys125 residue by Rsp5p and modified by Lys 63-linked ubiquitin chains. In contrast to the conclusions from prior reports, we demonstrated that, as noticed for most other multivesicular body cargoes, Sna3p ubiquitylation is required for its multivesicular body sorting. Sna4p is localized to the vacuolar membrane and interior. Sna4p contains an acidic di-leucine motif, that could be a sorting signal specific for AP-3 dependent pathway directing Sna4p to the vacuolar membrane. In apm3∆ cells, where µ subunit of the AP-3 complex is deleted, Sna4p is missorted to the vacuolar interior. Strikingly, this localization is different from localization of markers of AP-3 dependent pathway. This dissimilarity indicates that Sna4p possesses an additional characteristic, absent in other AP-3 cargoes, driving it to the vacuolar interior. In this study we have shown that the acidic di-leucine motif is indeed the sorting signal of Sna4p to the vacuolar membrane through the AP-3 dependent pathway, and that a part of Sna4p is targeted to the vacuole lumen via the multivesicular body pathway. The ability to enter multivesicular bodies is linked to the c-terminal PPPY sequence of Sna4p. Sna4p interacts with Rsp5p via this PY motif, resulting in Sna4p ubiquitylation on its lysine 128 and incorporation into the multivesicular bodies. Thus, Sna4p possesses two functional sorting signals which allow it to use two different pathways directing the protein to the vacuole.