The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket.

The tumour suppressor p53 is a key regulatory protein that prevents proliferation of damaged cells. Under unperturbed conditions, the ubiquitin ligase murine double minute 2 (MDM2) mediates p53 ubiquitination and further degradation by the proteasome. In consequence p53 is present at low levels, but becomes rapidly stabilised and activated in response to a variety of stimuli, such as DNA damage or virus infection. P53 responds to these diverse stresses to regulate the expression of many target genes that induce cell cycle arrest, DNA repair, or apoptosis. The attenuation of p53 interaction with MDM2 is maintained by enzymes catalysing p53 post-translational modifications such as phosphorylation. Casein kinase 1 α (CK1α) is one such enzyme; it stimulates p53 after DNA virus infection. Surprisingly depletion of CK1α using small interfering RNA or inhibition using a CK1 kinase inhibitor activated the transcription factor p53, indicating that p53 steady-state level is controlled by CK1α. Disrupting MDM2-p53 interaction using small molecule Nutlin-3 displayed similar pharmacological properties to the CK1 inhibitor on p53, indicating that the MDM2-CK1α complex co-regulates p53 stability. Indeed co-immunoprecipitation of endogenous CK1α with MDM2 occurred in undamaged cells. CK1α was shown in vitro to directly bind to and phosphorylate MDM2. Therefore it appears that CK1α must be recruited into specific complexes under different conditions, which can influence its substrate selectivity and explain its dual role on the p53 pathway. Apart from CK1, there are few other kinases whose action can directly contribute to the inhibition of p53. A novel pyrazolo-pyridine analogue showing dual activity against CK1 and Checkpoint kinase 1 led to increased p53 activation. These data highlighted the potential value of dual kinase inhibitors as therapeutics in cancer. The dominant protein-protein interface that stabilises the MDM2-CK1α complex was mapped using a peptide-based approach. One CK1α peptide bound strongly to MDM2, it specifically disrupted the protein-protein interaction, and its transfection was able to reduce cancer cell growth. A peptide phage display approach was finally combined with Next-Generation Sequencing to define the change in MDM2 binding motifs when the CK1α peptide or Nutlin-3 is bound, compared to ligand-free MDM2, and thus will help to understand protein-protein interaction network re-wirings which led to cell growth inhibition.

Universiẗat, Diss.--Stuttgart, 2001.

No description available.

The tumour suppressor p53 is essential for genome stability and loss of its function can lead to human cancer. The functional roles of p53 are regulated by a variety of mechanisms, some of which are not well understood. However, the murine double minute 2 (MDM2) protein, a major negative regulator of p53, has been found to be overexpressed in many human cancer cell lines in which p53 was not mutated; thus establishing MDM2 as a target for cancer therapeutics. MDM2 is defined as both an oncoprotein and an E3-ubiquitin ligase; its interactions with p53 are controlled through multiple domains, providing different possible pathways to inhibit MDM2 and therefore reactivate p53 function. Previous work undertaken in the Ball laboratory has shown that the MDM2 RING domain plays a critical role in p53 ubiquitination; thus screening for modulation of its activity by small molecules could provide new leads for the inhibition of the E3 ligase activity of MDM2. The MDM2 RING domain was cloned, expressed and purified so that it could be studied using a series of in vitro experiments. The generation of a library of short (12-mer) peptides as potential inhibitors of the MDM2 RING domain was investigated using phage display against His-tagged RING protein to screen the peptide ligands. In order to study the specificity of these peptides towards MDM2 (res. 396-491 and 396-479) compared with MDM4 and BRCA1, the MDM4 RING domain (res. 395-490 and 395-478) and BRCA1 (res. 1-304) domain were expressed and purified for further characterisation. A small selection of peptides was isolated and their binding affinity and activity as MDM2 inhibitors evaluated by in vitro ELISA, affinity chromatography and ubiquitination assays. One peptide in particular, KCCYFETHMPRH, was found to bind to MDM2 and was able to inhibit MDM2-mediated ubiquitination of p53 in vitro. Preliminary optimisation of this peptide by alanine scan revealed a peptide with a 2-fold increased potency. Since peptides provide comparatively weak therapeutic leads due to a combination of poor cellular uptake and susceptibility to cleavage by proteases, cyclic peptidomimetics based upon this lead were developed using side-chain to side-chain cyclisation. These peptidomimetics were successfully generated by the synthesis and incorporation of novel N-propargylated glycine and N-azidoalkyl glycine building blocks into a peptide sequence by Solid Phase Peptide Synthesis (SPPS). Following a Copper-catalysed Azide-Alkyne Cycloaddition (CuAAC) on solid phase or in solution, these peptoid-peptide hybrids were isolated, purified and characterised.

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. In the first part of the thesis, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by crossing the conditional Mdm4 and Mdm2 alleles to an erythroid-specific-cre (EpoRGFP-Cre ) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, interestingly, Mdm4 only contributes to p53 regulation at a specific phase of the differientation program. Moreover, a large body of evidence indicates that aberrant expression of either MDM2 or MDM4 impairs p53 tumor suppression function and consequently favors tumor formation. Overexpression of MDM2 was observed in 10% of 8000 human cancers from various sites, including lung or stomach, and MDM4 was found amplified and/or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach and breast cancers. Remarkably, selective MDM4 amplification occurs in about 65% of human retinoblastomas. In contrast, MDM2 amplifications are relatively rare (about 5%) in retinoblastomas, indicating that depending on the tumor context (cell type, initiating oncogene, …), MDM4, rather than MDM2, overexpression might be selected for as a more efficient mean of suppression of p53 function. As part of a large effort to better understand why different cell types require distinct combinations of mutations to form tumours, we will examine the molecular basis for selective up-regulation of Mdm4 in retinoblastomas. In this context, we have successfully generated 2 conditional transgenic mouse lines expressing either mycMdm2 or mycMdm4 driven by the PCAGGs promoters in the ROSA26 locus. Since a cassette containing a floxed transcriptional stop element is inserted upstream of the transgenes, we can achieve tissue-specific expression and spatio-temporal regulation of the transgenes by using different Cre and CreER. By the use of N-terminal myc-tag fused with the transgenes, we are able to compare the expression levels of the transgenes. Finally, due to C-terminal IRES-GFP element, we can easily identify transgene expressing cells. One of our aims is to use this Mdm4 conditional transgenic mouse line as the first, non-chimeric, mouse model of retinoblastoma that can be used as an appropriate preclinical model to improve treatment of this disease.

p53 is a critical tumour suppressor which acts to repair or remove abnormal cells and thus prevent cancer. Aberrant function of p53 is therefore a critical step in tumourigenesis and p53 is mutated in half of all cancers. Mutation of p53 leads to both a loss of normal wildtype function as well as the gain of oncogenic function. p53 is considered to be a promising therapeutic target and therapeutic strategies for targeting of the p53 pathway include: 1. Activation of wild-type p53 (wtp53) protein function, 2. Refolding of mutant p53 (mtp53) into the wtp53 conformation, 3. Reduction of mtp53 protein levels. In this work a number of small molecule screening assays were used to identify potentially novel regulators of both wtp53 and mtp53. Screening of a protein kinase inhibitor library for small molecules which can stimulate wtp53 activity identified the GSK3 pathway and a CDK pathway as dominant suppressors of wtp53 function. Screening of the library for inhibitors which reduce mtp53 protein levels led to the identification of two IKKβ inhibitors. The work then focused on investigating the effects of one of these compounds, IMD0354, on the mutant p53 pathway; with a specific focus on MDM2 as the most rapidly responding biomarker. IMD0354 is a well characterised inhibitor which has been shown to specifically inhibit IKKβ leading to the repression of the Nf-κB pathway. This study shows that IKKβ inhibition leads to the loss of a number of oncogenic proteins including mtp53, MDM2 and cyclin D. Mass-spectrometry based (ITRAQ) proteomic analysis was then employed to identify potential mediators and/or co-regulated factors in response to IKKβ-inhibition via IMD0354 treatment. This led to the identification of RPS3 as a potential negative regulator of MDM2 protein expression; the reduction in MDM2 protein in response to IMD0354 treatment is shown to be partially dependent on RPS3. Together this data has identified, using small molecule kinase inhibitor libraries: (i) dominant kinase signalling pathways that suppress wt-p53 and (ii) a dominant kinase signalling pathway that sustains expression of mutant p53 and MDM2 in cancer cell lines. This latter data supports further investigation to aid understanding of how the IKK signalling pathway cross-talks to the p53-MDM2 axis.

p53 is a key component of the protein network that regulates cell cycle progression and prevents cancer. Under non-stressed conditions, its activity is controlled by an autoregulatory feedback loop with MDM2 that maintains low levels of the p53 protein. However, in response to stress signals, p53 is triggered to become active. MDM2 has been reported to regulate p53 by a combination of mechanisms: ubiquitination using its E3-ligase capability, chaperone activity in an ATP-dependent manner and directly transrepressing p53. Because of MDM2's central role in the control of p53, it has been the target of intense drug development efforts. A family of small molecules, the Nutlins, can bind to an MDM2 pocket modulating the p53: MDM2 complex. This leads to p53 activation and growth inhibitory effects. The aim of our study was to analyse the interactome of endogenous MDM2 and to determine whether anti-cancer drugs, such as Nutlin-3, could stabilise or disrupt sets of MDM2 interactions in order to better understand the p53- dependent and independent functions of MDM2 as a signalling hub, as well as the p53-independent activity of Nutlin-3. Results show a remarkable difference in the sets of proteins found in MDM2 complexes in control and Nutlin-3 treated cells. Two proteins, TRIM25 and OTUB2, were selected from the output list for validation based on their known functions in the ubiquitin signalling network. Binding has been studied in detail and confirmed using both in cell and in vitro techniques. The data highlight potentially novel functions for MDM2 and provides insight into the on-target p53-independent activities of Nutlin-3. Additionally, and with the aim of blocking p53 ubiquitination by MDM2, I have developed probes that are able to inhibit the ubiquitylation of p53 in vitro.

No description available.

The tumour suppressor p53 has evolved a MDM2-dependent feedback loop that has a dual role as either a stimulator of p53 protein translation through mRNA binding or a stimulator of p53 protein degradation through the ubiquitin-proteasome system. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic drug-binding pocket and we have evaluated whether the lid of MDM2 is a physiological regulator of this dual function of MDM2. Deletion of this flexible pseudosubstrate motif inhibits MDM2 indicating that this peptide stretch can function as a positive regulatory motif. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2S17D) stabilizes the binding of MDM2 towards p53. Molecular modeling orientates the pseudo-substrate motif over a charged surface patch on the MDM2 surface at Arg97/Lys98 and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phosphomimetic mutation. Transient or inducible low level expression of MDM2WT can promote an increase in p53 protein steady-state levels whilst the expression of MDM2S17D in cells results in p53 protein de-stabilization. Phospho-specific antibodies to the MDM2 lid demonstrate two physiological conditions that alter lid phosphorylation: (i) lid hypo-phosphorylation occurs after DNA damage where p53 protein is stabilized and (ii) lid hyper-phosphorylation occurs at high cell density under conditions where p53 protein is de-stabilized. Expression of MDM2S17D in cells also de-stabilizes hyperubiquitinated mutant p53 under conditions where MDM2WT has no effect on mutant p53 protein degradation. The lid functions as a flexible regulatory motif whose phosphorylation switches MDM2 from a synthesis mode to a degradation mode with implications for defining the physiological signals that control the MDM2-p53 feedback regulatory loop.