Mutual dependence of MDM2 and MDMX in their functional inactivation of p53

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.     

MDMX overexpression prevents p53 activation by the MDM2 inhibitor Nutlin

The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.     

Targeting the conformational transitions of MDM2 and MDMX: Insights into key residues affecting p53 recognition

The oncogenic proteins MDM2 and MDMX have distinct and critical roles in the control of the activity of the p53 tumor suppressor protein. Recently, we have used spatial coarse graining simulations to analyze the conformational transitions manifest in the p53 recognition of MDM2 and MDMX. These conformational movements are different between MDM2 and MDMX and unveil the presence of conserved and nonconserved interactions in the p53 binding cleft that may be exploited in the design of selective and dual modulators of the oncogenic proteins. In this study, we investigate the conformational profiles of apo‐ and p53‐bound states of MDM2 and MDMX using molecular dynamic simulations along a time scale of 60 ns. The analysis of the trajectories is instrumental to discuss energetical and conformational aspects of p53 recognition and to point out specific key residues whose conformational shifts have crucial roles in affecting the apo‐ and p53‐bound states of MDM2 and MDMX. Among these, in particular, linear discriminant analyses identify diverse conformations of Y99/Y100 (MDMX/MDM2) as markers of the apo‐ and p53‐bound states of the oncogenic proteins. The results of this study shed further light on different p53 recognition in MDM2 and MDMX and may prove useful for the design and identification of new potent and selective synthetic modulators of p53‐MDM2/MDMX interactions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

On the interaction mechanisms of a p53 peptide and nutlin with the MDM2 and MDMX proteins: A Brownian dynamics study

The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53/nutlin interactions with MDM2 and MDMX and could potentially have a broader impact on anticancer drug optimization strategies.     

The MDMX acidic domain competes with the p53 transactivation domain for MDM2 N-terminal domain binding

The tumor suppressor protein p53 governs many cellular pathways to control genome integrity, metabolic homeostasis, and cell viability. The critical roles of p53 highlight the importance of proper control over p53 in maintaining normal cellular function, with the negative regulators MDM2 and MDMX playing central roles in regulating p53 activity. The interaction between p53 and either MDM2 or MDMX involves the p53 transactivation domain (p53TD) and the N-terminal domains (NTD) of MDM2 or MDMX. Recently, the acidic domain (AD) of MDMX was found to bind to its own NTD, inhibiting the p53-MDMX interaction. Given the established structural and functional similarity between the MDM2 and MDMX NTDs, we hypothesized that the MDMX AD would also directly bind to MDM2 NTD to inhibit p53-MDM2 interaction. Through solution-state nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that the MDMX AD can indeed directly interact with the MDM2 NTD and, as a result, can compete for p53 binding. The MDMX AD is thus able to serve as a regulatory domain to inhibit the MDM2-p53 interaction and may also play a direct role in p53 activation.     

MDM2 promotes ubiquitination and degradation of MDMX

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.     

Critical contribution of the MDM2 acidic domain to p53 ubiquitination

MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.     

Design of a novel MDM2 binding peptide based on the p53 family

p53 is a major tumor suppressor protein, that binds to, and is negatively regulated by MDM2. In tumors over expressing MDM2, p53 function can be rescued through the disruption of the MDM2-p53 interactions by small molecules and peptides. It is known that MDM2 also binds p73 but not p63, the two homologues of p53. We dissect the structural and energetic reasons underlying this discrimination and have identified a peptide that is intrinsically less helical than p53 and yet has a higher affinity for MDM2. The increased disorder has been introduced by localizing a cationic residue in between two anionic residues, imparting a degree of frustration to the system. In addition, the introduction of a bulkier hydrophobic group towards the centre of the peptide enables the peptide to adapt a bound conformation that on the one hand is most strained, and yet enables the peptide to straddle the largest surface of MDM2, amongst all the peptides. Computations also reveal that this peptide is a dual inhibitor, binding to MDMX. The computed affinity of the new peptide has been validated against MDM2 using fluorescence-based thermal shift assays.     

p53 inactivation by MDM2 and MDMX negative feedback loops in testicular germ cell tumors

Testicular germ cell tumors (TGCT) are unique in their excellent response to DNA-damaging chemotherapy. Mutation of p53 is rare in both untreated and relapsed TGCTs, suggesting that p53 fails to respond effectively against malignant transformation in germ cells. Previous studies implicated the presence of a poorly defined TGCT-specific mechanism of p53 inactivation. Here we show that disruption of p53-MDM2 binding using the MDM2-specific inhibitor Nutlin activates p53 in TGCT cells and is sufficient to induce strong apoptosis. Knockdown of MDMX cooperates with Nutlin to activate p53. Surprisingly, we found that p53 activation induced a two-fold increase in MDMX mRNA and protein expression in TGCT cells. A p53-responsive promoter is identified in MDMX intron 1 that contains a functional p53-binding site, suggesting that MDMX also functions as a negative feedback regulator of p53 in a cell line-dependent fashion. These findings suggest that MDM2 and MDMX are responsible for the functional inactivation of p53 in TGCT. Furthermore, TGCT cells are unique in having a strong apoptosis response to p53. Direct activation of p53 by targeting MDM2 and MDMX may provide a backup approach for the treatment of TGCTs resistant to DNA-damaging drugs.     

MDMX promotes proteasomal turnover of p21 at G1 and early S phases independently of, but in cooperation with, MDM2

We have shown previously that MDM2 promotes the degradation of the cyclin-dependent kinase inhibitor p21 through a ubiquitin-independent proteolytic pathway. Here we report that the MDM2 analog, MDMX, also displays a similar activity. MDMX directly bound to p21 and mediated its proteasomal degradation. Although the MDMX effect was independent of MDM2, they synergistically promoted p21 degradation when coexpressed in cells. This degradation appears to be mediated by the 26S proteasome, as MDMX and p21 bound to S2, one of the subunits of the 19S component of the 26S proteasome, in vivo. Conversely, knockdown of MDMX induced the level of endogenous p21 proteins that no longer cofractionated with 26S proteasome, resulting in G₁ arrest. The level of p21 was low at early S phase but markedly induced by knocking down either MDMX or MDM2 in human cells. Ablation of p21 rescued the G₁ arrest caused by double depletion of MDM2 and MDMX in p53-null cells. These results demonstrate that MDMX and MDM2 independently and cooperatively regulate the proteasome-mediated degradation of p21 at the G₁ and early S phases.     

Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans

MDM2, a ubiquitin E3-ligase of the RING family, has a key role in regulating p53 abundance. During normal non-stress conditions p53 is targeted for degradation by MDM2. MDM2 can also target itself and MDMX for degradation. MDMX is closely related to MDM2 but the RING domain of MDMX does not possess intrinsic E3-ligase activity. Instead, MDMX regulates p53 abundance by modulating the levels and activity of MDM2. Dimerization, mediated by the conserved C-terminal RING domains of both MDM2 and MDMX, is critical to this activity. Here we report the crystal structure of the MDM2/MDMX RING domain heterodimer and map residues required for functional interaction with the E2 (UbcH5b). In both MDM2 and MDMX residues C-terminal to the RING domain have a key role in dimer formation. In addition we show that these residues are part of an extended surface that is essential for ubiquitylation in trans. This study provides a molecular basis for understanding how heterodimer formation leads to stabilization of MDM2, yet degradation of p53, and suggests novel targets for therapeutic intervention.     

NMR structure of a complex between MDM2 and a small molecule inhibitor

MDM2 is a regulator of cell growth processes that acts by binding to the tumor suppressor protein p53 and ultimately restraining its activity. While inactivation of p53 by mutation is commonly observed in human cancers, a substantial percentage of tumors express wild type p53. In many of these cases, MDM2 is overexpressed, and it is believed that suppression of MDM2 activity could yield therapeutic benefits. Therefore, we have been focusing on the p53-MDM2 interaction as the basis of a drug discovery program and have been able to develop a series of small molecule inhibitors. We herein report a high resolution NMR structure of a complex between the p53-binding domain of MDM2 and one of these inhibitors. The form of MDM2 utilized was an engineered hybrid between the human and Xenopus sequences, which provided a favorable combination of relevancy and stability. The inhibitor is found to bind in the same site as does a highly potent peptide fragment of p53. The inhibitor is able to successfully mimic the peptide by duplicating interactions in three subpockets normally made by amino acid sidechains, and by utilizing a scaffold that presents substituents with rigidity and spatial orientation comparable to that provided by the alpha helical backbone of the peptide. The structure also suggests opportunities for modifying the inhibitor to increase its potency.     

Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of ^17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or ^17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and ^17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and ^17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K_d values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-^25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.