Thesis (Ph. D.) in Biochemistry and Molecular Biology--University of Maine, 2001. / Includes vita. Includes bibliographical references (leaves 97-113).

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references (p. 99-107).

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / 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 April 27, 2009) Vita. Includes bibliographical references.

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.

Nervenzellen sind hochspezialisierte Zellen, die an Synapsen miteinander verbunden sind, was die Übertragung von neuronalen Informationen erlaubt. Die Entwicklung von Synapsen und die Informationsverarbeitung und Gedächtnisbildung bei reifen Synapsen erfordert eine dynamische Umorganisation von neuronalen Netzwerken. Das beinhaltet die Bildung und Entfernung von Synapsen, Umsatz von synaptischen Proteinen und die Veränderung und Anpassung von synaptischer Erregungsübertragung. U. a. Ubiquitinierung, als regulatorische, posttranslationale Modifikation von Proteinen, könnte eine entscheidende Rolle für solche komplexe, synaptische Umorganisationen spielen. Nedd4-1, eine HECT-Typ E3 Ubiquitin Ligase, reguliert und fördert die Entwicklung von Nervenzellfortsätzen durch die Ubiquitinierung von Rap2. Um die Bedeutung von Nedd4-abhänginger Ubiquitinierung im entwickelten Gehirn zu untersuchen, wurden Mausmodelle generiert und analysiert, in denen Nedd4-1 und dessen nächstes Homolog Nedd4-2, speziell in Nervenzellen ausgeschaltet wurde. Ich habe herausgefunden, dass Nedd4-1 und Nedd4-2 wichtige regulatorische Proteine für die neuronale Morphogenese und die synaptische Plastizität, insbesondere die Aufrechterhaltung von LTP, darstellen. Desweiteren habe ich festgestellt, dass Synaptopodin (SYNPO), ein Prolin-reiches, Aktin-assoziiertes Protein, von Nedd4-1 und Nedd4-2 in vitro ubiquitiniert wird. Dieses Ergebnis deutet daraufhin, dass SYNPO in dem Mechanismus eine Rolle spielt, durch den Nedd4-1 und Nedd4-2 LTP aufrechterhalten. Diese Studie wirft ein neues Licht auf die funktionelle Rolle von Nedd4-abhänginger Ubiquitinierung bei höheren Funktionen des Gehirns von Säugetieren sowie der neuronalen Entwicklung.

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

Selective protein degradation by the ubiquitin-proteasome system is essential for cellular homeostasis and the regulation of diverse biological processes. The selectivity of this system is imparted by hundreds of ubiquitin ligases that specifically recognise substrates and catalyse their ubiquitination, thereby targeting them for degradation. Among ubiquitin ligases, multisubunit cullin-RING ubiquitin ligases constitute the largest group. However, despite significant advances in understanding their assembly, regulation, and molecular architecture, the substrates and functions of most of them remain unknown. This thesis focuses on two ubiquitin ligases from the cullin-RING ubiquitin ligase 4 (CRL4) subfamily: CRL4DCAF4 and CRL4DCAF12 . To identify their candidate substrates and to address their biological roles, several different approaches have been employed. First, proteomic screening revealed a wide range of candidate substrates. Next, detailed characterisation of the identified interactions and exploration of the condition under which candidate substrates undergo degradation was performed. Finally, knockout human cell lines and mice with a targeted disruption of genes encoding DCAF4 and DCAF12 were generated to explore the physiological roles of CRL4DCAF4 and CRL4DCAF12 . In summary, the herein...

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 PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references