Drosophila Nedd4 (DNedd4), an E3 ubiquitin ligase, is known to be involved in neuromuscular (NM) synaptogenesis during embryogenesis. To further elucidate its mechanism and function in this process, two major splice isoforms, dNedd4 short (dNedd4S) and dNedd4 long (dNedd4L), were studied. My work shows that while dNedd4S positively regulates NM synaptogenesis, dNedd4L plays a negative role in this process. Unique regions in dNedd4L, including the N-terminal 66 amino acid-long sequence (but not the putative dAkt phosphorylation site) and the middle 159 amino acid-long sequence, as well as the catalytic site, are required for its negative function. I proposed one possible mechanism of dNedd4L acting as a negative regulator of dNedd4S. Results from my studies of the putative effect of dNedd4L on the catalytic activity of dNedd4S in vitro, as well as on the function of dNedd4S towards Comm in Drosophila S2 cells, did not support this mechanism.

Drosophila Nedd4 (DNedd4), an E3 ubiquitin ligase, is known to be involved in neuromuscular (NM) synaptogenesis during embryogenesis. To further elucidate its mechanism and function in this process, two major splice isoforms, dNedd4 short (dNedd4S) and dNedd4 long (dNedd4L), were studied. My work shows that while dNedd4S positively regulates NM synaptogenesis, dNedd4L plays a negative role in this process. Unique regions in dNedd4L, including the N-terminal 66 amino acid-long sequence (but not the putative dAkt phosphorylation site) and the middle 159 amino acid-long sequence, as well as the catalytic site, are required for its negative function. I proposed one possible mechanism of dNedd4L acting as a negative regulator of dNedd4S. Results from my studies of the putative effect of dNedd4L on the catalytic activity of dNedd4S in vitro, as well as on the function of dNedd4S towards Comm in Drosophila S2 cells, did not support this mechanism.

The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity. This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells. The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.

The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity. This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells. The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.

?4 phosphoprotein (also known as IGBP1) is a component of the mammalian target-of-rapamycin (mTOR) pathway that controls the initiation of translation and cell-cycle progression in response to nutrients and growth factors. Aberrant signaling of the mTOR pathway has been reported in many cancers. ?4 interacts with the catalytic subunit of protein phosphtase 2A (PP2Ac) to mediate the dephosphorylation of eukaryotic initiation factor 4E-binding protein1 (4E-BP1) and p70S6 kinase (p70S6K). Our laboratory has reported that EDD E3 ubiquitin ligase (EDD/UBR5) and poly(A)-binding protein (PABP) are novel binding partners of ?4 phosphoprotein. In the present study, the interaction of EDD and PABP with ?4 was confirmed in human MCF-7 breast cancer and African green monkey COS-1 kidney cell lines, using immunoprecipitation and immunoblotting (IP/IB) analysis. However, co-IP of total MCF-7 cell lysates with anti-EDD antibodies revealed that EDD does not physically interact with PP2Ac. Several ?4 deletion constructs, that contained either the N-terminal or C-terminal regions of ?4, were transfected into MCF-7 and COS-1 cells. Co-IP studies with anti-EDD and PABP antibodies revealed that EDD interacts with the C-terminal region of ?4 whereas PABP, like PP2Ac, binds to the N-terminal region. EDD and PABP were found to interact with ?4 in both quiescent and actively growing cells. EDD is known to ubiquitinate poly(A)-binding protein-interacting protein 2 (Paip2), targeting it for proteosomal degradation. Paip2 is an antagonist of PABP activity. When ?4 levels in MCF-7 cells were knocked down using small interfering RNA (siRNA), there was no effect on EDD protein levels. There was also no effect on Paip2 levels, indicating that ?4 is not involved in the EDD- mediated ubiquitination of Paip2. Knockdown of EDD gene expression by siRNA did not alter mono-ubiquitination of ?4, indicating that ?4 is not a substrate of EDD. However, knockdown of EDD gene expression decreased poly-ubiquitination of PP2Ac and increased the overall cellular levels of PP2Ac, suggesting PP2Ac as a novel substrate of EDD. The present study suggests a potential role for ?4 in PABP-mediated initiation of mRNA translation. Furthermore, this study suggests a role for EDD in regulating PP2Ac levels through its interaction with ?4. In summary, the ?4 partners EDD, PABP and PP2Ac interact at specific regions of ?4. PP2Ac, but not ?4, is a substrate of EDD. The interaction of PABP with ?4 suggests a potential role for ?4 in PABP-mediated initiation of mRNA translation.

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.

Shigella flexneri is a highly adapted pathogen that invades the host cytosol and causes bacillary dysentery. Shigella has evolved powerful countermeasures to disarm host defense mechanisms; amongst them a family of twelve bacterial E3 ubiquitin ligases (IpaH) that are structurally unrelated to eukaryotic enzymes. IpaH ligases are injected into the host cytosol via the bacterial type III secretion system (T3SS) to manipulate the host cell and counteract anti-bacterial defense pathways. My work demonstrated that IFN-induced guanylate-binding proteins (GBPs) are novel targets for IpaH9.8. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria unless they are ubiquitylated by IpaH9.8 and consequently degraded by the proteasome. IpaH9.8 targets GBP1, GBP2, and GBP4, thereby causing a transient poly-ubiquitin coat comprising K48 and K27-linked chains around S. flexneri, which leads to the proteasome-dependent destruction of existing GBP coats and the re-establishment of bacterial motility and cell-to-cell spread. So far, ubiquitylation of bacteria has mostly been associated with anti-bacterial autophagy or immune signaling. However, the ubiquitin coat assembled around intracellular Shigella by IpaH effectors, in particular IpaH9.8, serves a pro-bacterial function, the first observed so far. In addition, I characterized IpaH1.4 and IpaH2.5 for their ability to prevent NF-κB activation by targeting LUBAC. I found that IpaH1.4 specifically binds the LUBAC component HOIP and mediates its proteasomal degradation, thus abolishing linear ubiquitylation of bacteria and consecutive NF-κB activation via NEMO and autophagy induction via optineurin. Lastly, I identified novel potential ubiquitylation targets for IpaH effectors in human cells using a mass spectrometry-based approach. The resulting IpaH interactome presents the groundwork for further investigations and will help to identify potentially unknown cellular defense mechanisms that are antagonized by Shigella flexneri.

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Madoz-Gurpide et al. recently observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF-β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. The results of chapter one report the existence of a nuclear variant of Bmp4. nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Chapter 2 and 3 further report that nBmp4 interacts with several subunits in the SCF E3 ubiquitin ligase, namely two Regulator of Cullins (ROC) proteins, five Cullin proteins, and two F-box proteins. Due to the known role of the SCF E3 ubiquitin ligase in regulating the cell cycle, the effect of nBmp4 on cell cycle progression was analyzed and the results show that nBmp4 affects the cell cycle by causing cells to accumulate in G0/G1. The association of nBmp4 and the SCF E3 ubiquitin ligase components and the affect that nBmp4 has on the cell cycle suggest that nBmp4 functions in the nucleus by inhibiting the SCF E3 ubiquitin ligase from ubiquitinating target proteins that are involved in regulating cell cycle progression. Finally, the initial stages in the generation of an nBmp4 over-expression mouse are described. The results of this research clearly change the traditional paradigm that Bmp4 performs all of its functions via extracellular signaling and introduce the existence of a nuclear variant that is involved in cell cycle regulation.

Tumor susceptibility gene 101¡]TSG101¡^encodes an inactive ubiquitin conjugating E2 enzyme implicated in regulation of protein sorting, vesicular trafficking, transcription activation of nuclear receptor, cell growth and differentiation. Previous studies showed that TSG101 can be mono- or poly- ubiquitinated, which is relevant to its functional status. There are seven Lysine (K) sites, K6, K11, K27, K29, K33, K48 and K63, on ubiquitin (Ub). Polyubiquitination using different Ub K sites confers differential function for protein degradation, DNA damage repair, endocytosis and protein sorting. AIP4 E3 ubiquitin ligase modifies its substrates involved in erythroid and lymphoid lineage differentiation and the associated immune responses. Mutation in AIP4 gene resolves in multisystemic autoimmune disease. TSG101 was recently shown to be a molecular checkpoint for T cell receptor downregulation. Here we investigate the ubiqutination status of TSG101. The ubiquitin-conjugated protein in lysate of cells co-transfected with pHA-TSG101 and His-tagged wild type Ub or each K site mutant ubiquitin expression plasmids was purified on nickel beads and then subjected to western blotting using antibodies against HA-TSG101 or His-tag. The results showed that K series mutant had differential effect on the steady-state of HA-TSG101. Proteasome inhibitor could alleviate its degradation especially in the K63 ubiquitin expression group, implying K63 ubiquitination E3 ligase is critical in maintaining HA-TSG101 level. Our coimmunoprecipitation result demonstrated the interaction between AIP4 and HA-TSG101, implying that TSG101 might be a substrate for AIP4. The ectopic overexpression of AIP4 increased the amount of HA-TSG101 in an E3 ligase activity depended manner. Taken together, these results indicated that AIP4 activity mediating Ub K63 modification might be critical for regulating cellular TSG101 protein level. Further experiment should clarify this issue.

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