Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 77-82.

Stuttgart, Univ., Diss., 2001.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 94-133).

Universiẗat, Diss., 2002--München.

München, Techn. Univ., Diss., 2002.

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.

The MDM2 protein regulates the tumour suppressor protein p53, acting as its chaperone, regulating its translation and targeting p53 for degradation by the 26s proteasome via its E3 ligase activity. The E3 ligase activity of MDM2 is dependent on its C-terminal RING domain. E3 ligases containing a RING domain are traditionally thought to catalyse the transfer of ubiquitin from their conjugating enzyme (E2) partner to the target protein, in the final step of the ubiquitination cascade. Various E2 enzymes have been shown to interact with their partner E3 ligases, yet evidence for the interaction between MDM2 and its partner E2, UbcH5α has not yet been shown. It has been reported that the reason for this lack of evidence is that the interaction between the two is highly unstable. Here I show that MDM2 forms a stable isolatable interaction with UbcH5α, the C-terminal tail of MDM2 is not necessary for this interaction. Although RING E3 ligases were not previously thought to interact with ubiquitin, preliminary evidence is emerging that suggests that this interaction is possible indeed I show that MDM2 and ubiquitin form a stable complex. I demonstrate that UbcH5α and ubiquitin both interact with the RING of MDM2, specifically the 20 most C-terminal amino acids of MDM2. My results show that both these proteins can bind this region of the RING simultaneously. I also highlight specific residues including tyrosine 489 and arginine 479 important for UbcH5α and ubiquitin binding respectively and the negative affect that these mutations have on the E3 ligase activity of MDM2 towards p53. Furthermore I show by limited proteolysis and hydrogen deuterium exchange that UbcH5α can be allosterically activated by MDM2. A novel peptide phage display technique linked to next generation sequencing was developed to further confirm an allosteric change and demonstrates that UbcH5α has different binding specificity for peptides when in a free or ligand bound conformation. MDM2 is a popular target for cancer therapeutics due to its dysregulation throughout many cancer types, including 30% of soft tissue sarcomas. Dissecting the mechanism of MDM2 function is an important step in identifying specific drugable interfaces on MDM2 and its interacting partners so that effective therapeutics can be designed.

Ubiquitin (Ub) ligases are important regulatory and signalling molecules, which are involved in majority of cellular processes such as differentiation, DNA repair, and regulation of energetic metabolism or immune response. E3 Ubiquitin ligases are also responsible for pathophysiological changes in the organism and their activity is associated with many human diseases including cancers. This makes E3 Ubiquitin ligases to be new diagnostic markers and interesting pharmaceutical targets. Based on previous studies, these enzymes evince very specific expression in the level of tissues or cell populations. Determination of this specific expression is important for a better understanding of their biological function. In this diploma thesis we systematically screened presence of 370 genes of E3-Ub ligases in gastrointestinal tract under physiological conditions and during acute inflammatory damage of distal colon. Obtained data allowed us to select genes, which can play important role in homeostasis as well as pathophysiology and regeneration of gastrointestinal tract. The screening was based on the expression profiling using qPCR, followed by in situ hybridization to determine the exact localization of the gene expression within tissues. From qPCR analysis was predicted hundred thirty seven candidates for...

TRIM7 is a member of the TRIM (tripartite motif-containing) protein superfamily. This family has been implicated in many disorders such as genetic diseases, neurological diseases, and cancers. Little is known about the function of TRIM7 except that it interacts with glycogenin and may regulate glycogen biosynthesis. Recently, a yeast two-hybrid protein-protein interaction screen revealed the binding of TRIM7 to Sin1, a protein found in a complex with the mammalian target of rapamycin (mTOR) protein kinase. mTOR can form two complexes, mTORC1 and mTORC2, which are important for cell growth, differentiation, and survival. Sin1 is a core component of mTORC2 and is critical for mTORC2 stability and activity. It was confirmed by co-immunoprecipitation that TRIM7 associates with Sin1 and mTOR in cultured mammalian cells. Furthermore, it was demonstrated that TRIM7 is a phosphoprotein, although it was not directly targeted by mTOR in vitro. Similar to some other TRIM family proteins, it was demonstrated that TRIM7 has a ubiquitin E3 ligase function allowing it to autoubiquitinate both in vitro and in cells. The autoubiquitination of TRIM7 was dependent on its RING domain. Further characterization of TRIM7 indicated that it can both homo-oligomerise as well as hetero-oligomerise with other members of its sub-class of TRIM proteins and that it co-localises with them into discrete cytoplasmic loci. To determine the cellular function of TRIM7, a stable cell line expressing an shRNA directed against TRIM7 was generated. Successful knock down of TRIM7 was achieved and this led to an increase in the protein levels of components of the mTORC2 complex, including Sin1. This coincided with an increase in cell proliferation. In conclusion, this research identifies a novel role for TRIM7 as a ubiquitin ligase involved in regulating cell proliferation and provides a potential link between TRIM7 and the mTOR pathway, a major transducer of proliferative and cell survival signals.