MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene. MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the ubiquitination (and degradation) of p53. Several proteins are responsible for the ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential for ubiquitination, the protein-protein recognition site is a potential drug target. Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues 386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these results showed that the MDM2RING construct formed a concentration-dependent oligomeric structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations. Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified. Various biophysical techniques were used to study its interaction with MDM2 as well as with potential small molecule inhibitors as in principle, small molecules which disrupt the interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53. The dimerisation of MDM2 is important for its E3 activity and the C8-binding site potentially provides a second druggable site. In this work, peptide 9, which has the same sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2 E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and electrospray mass spectrometry were used to investigate the interaction between two inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to search for small molecules which can affect MDM2-dependent ubiquitination. Three potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were identified using our virtual screening database-mining and docking programs that were shown to affect MDM2-dependent ubiquitination of p53. In terms of understanding the complex biochemical mechanism of MDM2 this work provides two interesting and functionally relevant observations: (i) the MDM2 RING△C construct is a dimer as this would not be expected form the existing studies, and has less E3 ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding site enhanced E3 ligase activity. One model to explain these observations is that binding of small molecule activators family to the RING induces a change in the conformation of the Cterminal tail residues which may enhance E2 binding.

The effect of structuring, in the form of magnetic or density inhomogeneities, on the magnetohydrodynamic (mhd) waves of an infinite plasma is investigated. The appropriate dispersion formulae, in both Cartesian and cylindrical polar coordinate geometries, are derived. The main properties of the allowable modes in structured plasmas are described, particularly those featuring in a slender inhomogeneity. The inclusion of non-adiabatic effects is examined, specifically for a thermally dissipative, unstratified, finite structure and for a slender inhomogeneity in a stratified medium. The dissipative time scales of slender structures are shown to have a dependence on the Peclet number. Growth factors appropriate to these time scales for the overstable motions of a thermally dissipative, Boussinesq fluid are derived. For the linear analysis of a slender structure it is shown that the dispersive nature of the waves is deducible from the simplified one-dimensional equations. The analysis is extended, for slender structures, to nonlinear motions and the governing equation representing an effective balance between nonlinear, dispersive and dissipative effects, the Benjamin-Ono-Burgers equation, is established. The solutions of this equation are considered and, for weakly-dissipative systems, are shown to be slowly decaying solitons. The importance, in the context of group velocity, of the dispersive nature of waves in ducted structures is discussed and analogies are made with other ducted waves, for example, the Love waves of seismology. It is suggested that the behaviour of such waves, following an impulse, may account for the range of oscillatory behaviour, the quasi-periodic and short time scales, observed in both the solar corona and Earth's magnetosphere. Density variations across a structure and the structure's curvature, with possible applications to coronal loops, are also considered. Further suggestions for possibly identifying some of the theoretical results with observed behaviour in sunspots, chromospheric fibrils and spicules are also made.

In his ethical treatises Aristotle offers a rich account of those conditions that render people’s behaviour involuntary, and defines voluntariness on the basis of the absence of these conditions. This dissertation has two aims. One is to offer an account of the significance of the notions of involuntariness and voluntariness for Aristotle’s ethical project that satisfactorily explains why he deems it necessary to discuss these notions in his Ethics. My own account of the significance of these notions for Aristotle’s Ethics emerges from my arguments against the two most influential views concerning this significance: I argue that Aristotle’s concern with voluntariness in his Ethics is not (primarily) shaped by a concern with accountability, i.e. with those conditions under which fully mature and healthy rational agents are held accountable or answerable for their actions; nor is it (primarily) shaped by a concern with the conditioning of pain-responsive agents for the sake of socially useful ends that are not, intrinsically, their own. Rather, his concern is with reason-responsive agents (which are not morally accountable agents, nor merely pain-responsive agents) and the conditions for attributing ethically significant behaviour to them. This is what I call ‘ethical ascription’. The second aim of this dissertation is to provide a comprehensive account of those conditions that defeat the ascription of ethically significant pieces of behaviour to reason-responsive agents, and to show the distinctiveness of Aristotle’s views on the nature of these conditions. The conclusions I arrive at in this respect are shaped by the notion of ethical ascription that I develop as a way of reaching the first aim.

The ubiquitylation cascade regulates multiple cellular functions and is involved in numerous diseases. The distinct transfer cascade, involving E1-E2-E3 enzymes, serves as a promising target for drug development. However, E3 ligases (E3s) represent an important class of enzymes yet there are currently no effective tools for profiling their activity. Herein, a new class of E3 activity-based probe (ABP) is presented which is built by re-engineering ubiquitin (Ub)-charged E2 conjugating enzymes. The utility of these probes has been demonstrated by the rapid dissection of the activation determinants of the RING-Between-RING E3 (RBR) E3, Parkin. Furthermore, biotin-E3 ABPs allow us to systematically discover and dissect the E3 activities of a broad spectrum of E3s that are associated with different diseases. By interfacing the ABPs with mass spectrometry, we establish an activity based protein profiling (ABPP) system and apply it to uncover a new class of E3. We show that MYCBP2 is an E3 ligase with a novel mechanism of action that ubiquitylates threonine residues. MYCBP2 contains a RING domain, that recruits the ubiquitin-loaded E2, and a novel Zn-binding fold that contains two catalytic cysteine residues which relay the Ub to substrate via two thioester intermediates (RING-Cys-Relay, RCR). This discovery demonstrates the power and potential of our E3 activity based protein profiling (ABPP) system.

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.

<p>The Anaphase Promoting Complex/Cyclosome (APC/C) is a RING E3 ligase that plays essential roles both within and outside of the cell cycle. At the onset of anaphase, the APC/C targets cyclin B and securin for degradation, initiating chromosome separation and mitotic exit. Regulation of APC/C activity is critical for a functional cell cycle, and this is largely mediated by cytostatic factor (CSF) activity and the Spindle Assembly Checkpoint (SAC). </p> <p>Prior to fertilization, vertebrate eggs are arrested in metaphase of meiosis II by CSF activity, a key component of which is the APC/C inhibitor Emi2. Although the roles and regulation of Emi2 in maintaining CSF arrest have been extensively studied, its function during the oocyte maturation process, especially at the meiosis I to meiosis II (MI-MII) transition, was not well understood. Studies presented in this dissertation characterize an Emi2-mediated auto-inhibitory loop of the APC/C that provides the molecular basis of a critical biochemical event during the MI-MII transition--the partial degradation of cyclin B. In brief, phosphorylation of the Emi2 N-terminus by Cdc2/cyclin B targets it for proteasomal degradation in meiosis I (MI). During anaphase of MI, the APC/C triggers its own inactivation by degrading cyclin B, therefore stabilizing its inhibitor, Emi2. The timely inactivation of APC/C activity prevents the complete inactivation of Cdc2 kinase, which is crucial for prohibiting S phase onset and parthenogenetic activation of the oocytes.</p> <p>To better understand the regulation of the APC/C, a number of the studies presented here are aimed at identifying the mechanism for Emi2 inhibition of the APC/C. Many APC/C inhibitors have been reported to function as "pseudosubstrates", which inhibit the APC/C by preventing substrate binding. After carefully examining the ubiquitin reactions mediated by the APC/C in vitro, we have found that it is the last step in the ubiquitylation process, where ubiquitin is transferred from a charged E2 to the substrate, that is targeted by Emi2. In addition, biochemical studies have also revealed that Emi2 itself has RING-dependent ligase activity and this activity enables it to inhibit the APC/C in a sub-stoichiometrical manner. </p> <p>Although the ultimate goal for both CSF activity and the SAC signaling pathway is APC/C inhibition, a much more complicated regulatory network is known to control SAC. Previous researches in our lab have identified Xnf7 to be an APC/C inhibitor that is required for the SAC pathway in Xenopus egg extract. In an effort to characterize the human Xnf7 homolog, we have found that Trim39, a protein that has been implicated in apoptosis regulation, is required for the SAC pathway in RPE cells. Like Emi2, both Xnf7 and Trim39 are RING E3 ligases whose activity is essential for their function. Interestingly, the ligase activity of both proteins appears to be regulated by the checkpoint. While we continue to characterize the roles and regulation of both Trim39 and Xnf7 in the SAC, future investigations into the mechanisms that underlie APC/C inhibition by all the three E3 ligases--Emi2, Xnf7 and Trim39--would be of great interest.</p> / Dissertation

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?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 covalent modification of proteins with a suite of molecular tags, a process termed post-translational modification, is a powerful means to enhance the proteomic complexity of an organism far beyond that which is directly encoded by its genome. A particularly widespread form of modification involves the conjugation of the protein ubiquitin to specified substrates, which serves to regulate numerous cellular processes. The mechanism of ubiquitin conjugation, known as ubiquitylation, requires E3 ubiquitin ligases that specify and recruit substrate proteins for ubiquitin conjugation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. This thesis describes the dimeric structure of the E3 ligase adaptor protein SPOP in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. Here, it is demonstrated that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. These results reveal a mechanism through which adaptor protein self-assembly may provide a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.