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The ubiquitin pathway is a highly conserved post-translational modification system best characterised for its roles in protein degradation and intracellular trafficking and is involved in a diverse spectrum of cellular processes. Ubiquitylation is opposed by deubiquitylating enzymes (Dubs), and the ubiquitin specific peptidase (USP) class of Dubs remove ubiquitin from specific substrates, thereby affecting protein fate. USPs exhibit broad sequence diversity except over their catalytic cores and it has been suggested that this sequence variation constitutes their individual substrate-specific binding sites. Fat Facets in Mouse (Fam) is a developmentally regulated USP whose function is crucial for mouse pre-implantation development. Fam is expressed in a complex fashion throughout development in a number of diverse tissue types and time points, well beyond its critical role in the early embryo. Fam's orthologue in fly, Fat Facets (faf) is also developmentally regulated and is required for both drosophila eye and syncytial stage development. Given the strengths of the zebrafish system as a developmental tool, the zebrafish orthologue of Fam, usp9 was identified and found to be highly conserved. Analysis of its expression pattern found considerable overlap with the published mouse patterns. Given the similarities between the mouse and zebrafish systems, a series of cross-species experiments were conducted to determine whether exogenous expression of highly conserved regions of FAM, could cause dominant-negative phenotypes in developing zebrafish embryos. Outside of the catalytic core, FAM's large N and C-terminal extensions consist of novel sequence bearing no similarity to any known domain. To delineate FAM domains, fulllength FAM was expressed in insect cells and subjected to partial proteolysis. Combining this data with recent structural predictions and computer analyses of the FAM sequence, four FAM domains were characterised with the first domain containing three possible subdomains. The predominant helical nature of the N and C-terminal extensions of FAM were predicted to form scaffolding structures, well suited to protein binding. / Thesis (M.Sc.)-- University of Adelaide, School of Molecular and Biomedical Science, 2006.

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Ubiquitin (Ub) is a small, 76 amino acid, and post-translational modification (PTM) protein in eukaryotes. Modification of a substrate protein via the covalent attachment of the C-terminal glycine of Ub to the ε-amino group of lysine residues in a substrate is termed ubiquitination. Unlike, other PTM proteins, Ub can form polyUb chains at one or more of its seven lysine residues. (K6, K11, K27, K29, K33, K48, and K68). The consequence of these different polymerization sites is altered biological response with different polyUb linkages conferring different fates to target proteins. Unfortunately, the study of these chains have been limited by the inability to generate homogeneous polyUbs chains linked at known lysine residues. Furthermore, a three step enzymatic cascade consisting of activating-enzymes (E1s), conjugating enzymes (E2s), and ligase enzymes (E3s) tightly controls this modification. In response, our laboratory has developed a system that creates polyUb chains through bacterial expression and "synthetic" building blocks. Now, the main questions are what do these chains interact with in the cell and how do these interactions mediate biological responses? In an attempt to answer these questions, this dissertation looks at different molecular techniques created to capture the transient interactions of monoUb and diUb probes with Ub substrates, such as, ubiquitin binding domains (UBDs) and conjugating E2 enzymes. One molecular technique focuses on the use of incorporating a genetically encoded, photo-crosslinker, p-Benzoyl-L-phenylalanine (pBpa) into diUb probes to capture their interaction with UBDs. This sets the foundation for understanding Ub’s cellular signaling recognition of UBDs. Another technique is creating diUb probes that contain lysine derivatives, Nε-L-Thiaprolyl-L-lysine (ThzK) or Nε-L-Cysteinyl-L-lysine (CysK), and can form a disulfide bonds with E2 enzymes to capture their complex, opening an opportunity to understand mechanistically the role E2 enzymes have with polyUb chain formation. Herein, these techniques are established to help unravel the complexity of Ub signaling.

Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 159-179).

The ubiquitination of proteins controls a multitude of physiological processes. This versatility of ubiquitin as a molecular signal arises from the diverse ways by which it can be attached to target proteins. Different ubiquitination patterns are then translated into different downstream consequences. Due to the enormous complexity of possible ubiquitin modifications, the ubiquitination machinery must be highly specific and tightly controlled. Ubiquitination proceeds through an enzymatic cascade, the last step of which is catalyzed by the E3 enzyme family. E3 enzymes are the crucial regulators since they dictate the specificity of substrate selection and modification. Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity, regulation and specificity in this crucial ligase are incompletely understood. One aim of this study was to unravel the role of the a1’-helix N-terminal to the HECT domain that was found to be a key element mediating regulation and oligomerization in other HECT ligases. I found that most N-terminally extended HECT domain constructs were insoluble when expressed in E. coli, indicating that additional regions N-terminal to the tested fragments may be essential to protect this highly hydrophobic helix from causing aggregation. Another question addressed in this study was how E6AP builds ubiquitin chains. Using single-turnover experiments, I showed that ubiquitin-loaded E6AP is unable to transfer an additional ubiquitin molecule onto a stably linked ubiquitin-E6AP complex. This indicates that E6AP cannot assemble chains on its active site and may instead follow a sequential addition mechanism in which one ubiquitin molecule is transferred at a time to the target protein. Using NMR spectroscopy and extensive mutational analyses, the determinants of ubiquitin recognition by the C-lobe of E6AP were unraveled and assigned to particular steps in the catalytic cycle. A functionally critical interface was identified that is specifically required during thioester formation between the C-terminus of ubiquitin and the ligase active site. This interface resembles the one utilized by NEDD4-type enzymes, suggesting a conserved ubiquitin binding mode across HECT ligases, independent of their linkage specificities. Moreover, I identified critical surface patches on ubiquitin and in the N- and C-terminal portions of the catalytic domain of E6AP that are important for the subsequent step of isopeptide bond formation. I also uncovered key determinants of the Lys48-linkage specificity of E6AP, both in the E6AP HECT domain and ubiquitin itself. This includes the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site, Lys48. It is thus tempting to speculate that ubiquitin linkage formation by E6AP is substrate-assisted. Taken together, my results improve our mechanistic understanding of the structure-function relationship between E6AP and ubiquitin, thus providing a basis for ultimately manipulating the functions of this HECT ligase for therapeutic applications. / Die Ubiquitinierung von Proteinen ist an nahezu jedem physiologischen Prozess beteiligt. Die Vielseitigkeit mit der Ubiquitin als molekulares Signal fungiert, rührt von den vielfältigen Möglichkeiten her, wie es an Zielproteine gebunden werden kann. Verschiedene Ubiquitinierungsmuster rufen unterschiedliche biologische Ereignisse hervor. Angesichts der enormen Komplexität möglicher Ubiquitinierungsmodifikationen muss die Ubiquitinierungs-maschinerie hochspezifisch und streng kontrolliert sein. Die Ubiquitinierung erfolgt über eine enzymatische Kaskade. Der letzte Schritt wird hierbei durch die Enzymfamilie der Ubiquitin-Ligasen katalysiert. Ubiquitin-Ligasen sind primär für die Spezifität in Substraterkennung und Ubiquitin-Kettenbildung verantwortlich. Misregulation der HECT-Ligase E6AP fördert die durch humane Papillomaviren induzierte Tumorentwicklung im Gebärmutterhals und ist mit zwei schweren neurologischen Krankheiten verbunden. Strukturelle Einzelheiten über den Mechanismus, die Regulation und die Spezifität dieser wichtigen Ligase sind jedoch weitgehend unbekannt. Für verschiedene HECT-Ligasen wurde gezeigt, dass die a1‘-Helix N-terminal zur HECT-Domäne ein Schlüsselelement für die Regulation und den Oligomerisierungszustand der Enzyme darstellt. In dieser Arbeit konnte gezeigt werden, dass die Helix eine wichtige Funktion für die Stabilität von E6AP erfüllt. Der Großteil N-terminal verlängerter, in E. coli exprimierter HECT-Domänen-Konstrukte war unlöslich, was darauf hindeutet, dass N-terminal gelegene Regionen hydrophobe Bereiche des Proteins vor Aggregation schützen. Eine weitere Fragestellung dieser Arbeit befasste sich mit dem Mechanismus der Ubiquitin-Kettenbildung durch E6AP. Mit ‘single-turnover‘-Experimenten konnte gezeigt werden, dass ein über einen Thioester gebundenes Ubiquitin von E6AP nicht auf einen stabil verknüpften Ubiquitin-E6AP-Komplex übertragen werden kann. Dies deutet daraufhin, dass E6AP keine Ketten auf dem katalytischen Cystein aufbauen kann und stattdessen einem sequentiellen Additionsmechanismus der Ubiquitin-Kettenbildung folgt. Mithilfe von NMR Spektroskopie und umfangreicher Mutagenese-Studien wurde eine Interaktion zwischen dem C-Lobe von E6AP und Ubiquitin gefunden, die während der Thioesterbildung zwischen dem C-Terminus von Ubiquitin und dem aktiven Zentrum von E6AP gebraucht wird. Diese Interaktionsfläche ähnelt derer der NEDD4-Familie, was auf einen konservierten Bindungsmodus der HECT-Ligasen an Ubiquitin im ersten Reaktionsschritt hindeutet, ungeachtet der jeweiligen Kettenspezifitäten. Verschiedene Oberflächen auf Ubiquitin und E6AP, sowohl auf dem C-Lobe als auch auf dem N-Lobe, konnten identifiziert werden, die für die Bildung einer Isopeptidbindung zwischen zwei Ubiquitin-Molekülen von Bedeutung sind. Neben dem C-Terminus von E6AP wurde eine hydrophile Oberfläche auf Ubiquitin in unmittelbarer Nähe zum Akzeptor Lys48 gefunden, die wichtig für die Lys48-spezifische Ubiquitin-Kettenbildung ist. Der Gedanke liegt nahe, dass die Ubiquitin-Kettenbildung durch E6AP über Substratunterstützte Katalyse verläuft. Zusammenfassend erweitern diese Ergebnisse maßgeblich unser Verständnis der Erkennung von Ubiquitin durch die HECT-Ligase E6AP und können möglicherweise dazu beitragen Wirkstoffe zu entwickeln, welche eine Fehlregulierung von E6AP ausgleichen können.

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