Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 158-177.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-104).

Ubiquitin is a highly conserved 76 amino acid protein which is a unique and versatile signalling molecule. Ubiquitin can be attached by an isopeptide bond between its C-terminal diglycine to a lysine residue of a target substrate. However, it can also bind to itself though one of its own seven lysine residues allowing the formation of different chain types. These chains act as signals for different pathways, such as DNA damage repair, and in particular lysine-48 chains signal for proteins to be degraded via the proteasome by the ubiquitin proteasome system (UPS). This allows cells to control the concentration of proteins which is important in triggering cellular events, such as cyclin levels in cell division. Whilst old and incorrect proteins need to be removed so they do not interfere with normal processes. In order to recognise and ubiquitinate substrates an enzyme cascade has evolved. Ubiquitin is transferred from an ubiquitin activation enzyme (E1) to an ubiquitin conjugating enzyme (E2). The E2 which along with a ubiquitin ligase (E3) ubiquitinates a specific substrate. Research has focused on the E3 ligases since they are responsible for identifying substrates. One important ligase is the anaphase promoting complex/cyclosome (APC/C) which is responsible for faithful segregation of chromosome during mitosis. Failure to regulate this process can lead to aneuploidy, one of the main causes of cancer. It is therefore important to understand the function and regulation of APC/C and the UPS. This work firstly shows that four S. pombe kinases, Ssp2, Ppk9, Kin1 and Chk1 all contain a kinase associated 1 (KA1) or KA2 domain which they use to interact specifically with APC/C when it contained an unphosphorylated form of a subunit called Cut9. Yeast two hybrid and native far Westerns demonstrated that the KA domains interact with the APC/C co activator Slp1. Phosphorylation assays showed that three of these kinases phosphorylated a ~30kDa band of the APC/C complex which was shown to be Mad2, an important subunit of the APC/C inhibitor complex the mitotic checkpoint complex (MCC). These finding suggest a new role for KA contain kinases as regulators of APC/C activity. Future studies to identify the residues of Mad2 which are phosphorylated by these kinases, as well as the binding site of Slp1 that the KA domains recognise, would provide a more detailed understanding of the molecular mechanisms involved in regulating APC/C activity. Secondly, this study investigated the role of the ubiquitin associated (UBA) domains in the S. pombe shuttle factor Rhp23. This protein can recognise the proteasome via an ubiquitin like (UBL) domain and ubiquitin chains via one of two UBA domains: an internal UBA1 and a C-terminal UBA2. To dissect the different functions of these two UBA domains point mutations were made that abolished the domains ability to recognise ubiquitin without altering the protein structure. The minimal domains and full length domains were tested in vitro and in vivo. These surprising results showed that the domains act differently in isolation when compared to the full length protein. They also demonstrate that the UBA1 domain is responsible for ubiquitin recognition in Rhp23, whilst the UBA2 domain appears to have little to no binding ability.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway.

No description available.

No description available.

The HECT E3 ubiquitin ligase Nedd4-1 was previously shown to regulate diverse processes such as cell and animal growth, insulin signaling, and lysosomal trafficking. To further elucidate the cellular functions of Nedd4-1, Nedd4-1 knockout mouse embryonic fibroblasts were characterized relative to their wild type counterparts. Immunofluorescence experiments revealed an altered lysosomal distribution in the knockout cells, although their lysosomal proteolytic function appeared normal. Transmission Electron Microscopy revealed striking morphological differences, especially regarding the lysosome and endoplasmic reticulum of the knockout cells. Another aspect of my studies examined the interaction between Nedd4-1 and Angiomotin (p130-AMOT), which involves the same motifs required to sequester transcriptional co-activators YAP and TAZ in the cytoplasm. To test either a competitive or non-competitive mode of binding, co-immunoprecipitation experiments involving p130-AMOT, the Nedd4 proteins, and YAP or TAZ were performed, with results not supporting a competitive mode of interaction. Overall, my results demonstrate new Nedd4-1 cellular functions.

Apoptose ist eine bestimmte Art des programmierten Zelltods. Dieser Prozess erfüllt zahlreiche wichtige physiologische Funktionen. Eine pathologische Dysregulation der Apoptose ist an der Entstehung etlicher Krankheiten beteiligt. Bei der Regulation der Apoptose nehmen die Bcl-2-Familienmitglieder und damit auch das anti-apoptotisches Familienmitglied A1 eine wichtige Stellung ein. Die Stabilität und anti-apoptotische Funktion von A1 wird über den Ubiquitin-Proteasomen-Weg reguliert. Hierbei ist das C-terminale Ende von A1 essentiell. Ausgangspunkt für diese Arbeit war die Hypothese, dass an den C-Terminus von A1 eine bisher unbekannte Ubiquitin-E3-Ligase bindet, verzweigte Ubiquitinketten an Lysinreste des A1-Proteins konjugiert und das Protein dadurch für den proteasomalen Abbau markiert. Durch Mutationen einzelner Lysinreste von A1 sollte untersucht werden, welche dieser Aminosäuren ubiquitinyliert werden. Damit sollte der molekulare Mechanismus, der hinter der Instabilität und der anti-apoptotischen Funktion steckt, weiter charakterisiert werden. In dieser Arbeit wurden insgesamt 11 Mutanten des A1-Proteins hergestellt, bei denen die 11 Lysinreste von A1 gruppenweise zu Argininresten (K-A1-Mutanten) ausge-tauscht wurden. Der Austausch von Lysin zu Arginin wurde gewählt, weil hierdurch die Ladung an der entsprechenden Position des Proteins gleich bleibt, während eine Konjugation von Ubiquitin an Arginin nicht möglich ist. Im Ergebnis zeigte sich, dass das A1-Protein nicht nur an einzelnen, ganz spezifischen Lysinen, sondern an allen oder doch zumindest den meisten seiner 11 Lysine ubiquitinyliert werden kann, denn es ergaben sich bei den K-A1-Mutanten keine signifikanten Unterschiede in Stärke oder Muster ihrer Ubiquitinylierung. Es müssen also nicht einige wenige Lysine notwendigerweise ubiquitinyliert werden, um A1 zu destabilisieren, sondern die Ubiquitinylierung ganz unterschiedlicher Lysine markiert das Protein für den proteasomalen Abbau. Auch hat der Verlust bestimmter Lysingruppen und damit potentieller Ubiquitinylierungsstellen keinen signifikanten Einfluss auf die anti-apoptotische Funktion des A1-Proteins. Zusammengefasst unterstützen die Ergebnisse die Hypothese, dass eine bisher nicht bekannte Ubiquitin-E3-Ligase sowohl die Stabilität als auch die anti-apoptotische Funktion von A1 reguliert, indem sie verzweigte Ubiquitinketten an (fast) alle Lysine des Protein anhängt und das Protein damit für den proteasomalen Abbau markiert. / Apoptosis is a certain kind of programmed cell death and plays an important role in many physiological processes. Pathological dysregulation of apoptosis is involved in the development of a number of diseases. The Bcl-2 family members and with it the anti-apoptotic family member A1 are important regulators of apoptosis by regulating the integrity of the mitochondria. A1 is a very unstable protein, its stability is regulated by the ubiquitin/proteasome pathway. Based on published work, the hypothesis was set up, that a so far unknown ubiquitin-E3-ligase binds to the C-terminal end of A1, attaches ubiquitin chains to lysine residues and thereby marks the protein for proteasomal degradation. The ubiquitinylation and the molecular mechanisms behind the instability and the anti-apoptotic capacity of A1 should be further characterized by mutating the lysine residues of the protein. For this paper altogether 11 mutants of the A1 protein were cloned. The 11 lysine residues of A1 were replaced in groups with arginine residues (K-A1 mutants). Arginine was chosen to keep the charge at that position in the protein but to inhibit ubiquitylation at this position. The experiments indicate that all or at least most of the lysine residues of the A1 protein can be ubiquitinylated, because no significant differences in ubiquitinylation of the K-A1 mutants compared to A1 wildtype protein could be observed. Thus, no specific lysine residues have to be necessarily ubiquitinylated to destabilize the A1 protein, but ubiquitinylation of different lysine residues can mark the protein for proteasomal degradation. Furthermore, the loss of certain lysines showed no significant influence on the anti-apoptotic capacity of A1. Altogether, the results support the hypothesis, that a so far unknown ubiquitin-E3-ligase attaches ubiquitin to most if not all lysine residues and thereby regulates the stability and the anti-apoptotic capacity of the protein.