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

Li, Chong; Pazgier, M.; Li, Changqing; Yuan, Weirong; Liu, Min; Wei, Gang; Lu, Weiyue; Lu, Wuyuan

doi:10.1016/j.jmb.2010.03.005

Cited by 124 publications

(198 citation statements)

References 58 publications

(102 reference statements)

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“…Uniformly 15 N-labeled or 15 N-and 13 C-labeled and unlabeled samples of WT and mutant versions of human p53TAD (residues 1-73) were prepared as previously described 12 . Samples of human Mdm2, corresponding to residues 17-125, were expressed using pGEX-6p-2 vectors in BL21(DE3) cells grown in M9 medium.…”

Section: Protein Purificationmentioning

confidence: 99%

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Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

et al. 2014

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Levels of residual structure in disordered interaction domains determine in vitro bindingaffinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage.These results establish that residual structure is an important determinant of signaling fidelity in cells.Intrinsically disordered protein domains often mediate protein-protein interactions that result in disorder-to-order transitions via coupled folding and binding reactions 1 . In addition, many disordered interaction domains exhibit defined levels of transient secondary structure resembling their complex-bound states when free in solution 2 . These levels of residual structure affect binding energies, also by reducing the loss of conformational entropy associated with disorderto-order transitions 3 . Accordingly, higher levels of residual structure in disordered interaction domains increase in vitro binding affinities 4,5 . Here we ask to what extent residual structure contributes to protein binding affinities in cells and whether engineered changes to residual structure affect protein function at the cellular level. To answer these questions, we designed p53 mutants with higher residual helicity within their disordered, N-terminal transactivation domains (TADs) and investigated their effects on cellular Mdm2 binding and p53's ability to induce 2 target gene expression and cell cycle arrest. p53 is activated by many forms of cellular stress, including DNA double-strand breaks (DSBs) and functions as a major tumor suppressor and cell cycle regulator 6 . In the absence of DNA damage, cellular p53 levels are kept low by targeted proteasomal degradation mediated by the E3 ubiquitin ligase Mdm2, which interacts with p53TAD and subsequently ubiquitinates p53's C-terminal regulatory domain 7 . Upon DNA damage, post-translational modifications of p53TAD and Mdm2, together with Mdm2 degradation, disrupt the p53-Mdm2 complex. This leads to p53 accumulation and the expression of p53 target genes that regulate DNA repair, cell cycle arrest, senescence or apoptosis 8,9 . One of these target genes is Mdm2, whose expression establishes a negative feedback loop that shapes cellular p53 dynamics and thereby controls cell fate decisions 10 .In its free form, p53TAD exists in equilibrium between disordered and partially helical conformations 11-13 , whereas residues 19-25 form a stable amphipathic α-helix in the Mdm2 complex 14 (Fig. 1a). To increase the binding affinity between p53 and Mdm2 without altering the binding interface, we designed p53TAD mutants with higher levels of residual helicity by mutating conserved proline residues flanking the Mdm2 binding site (i.e., Pro12, Pro13 or Pro27) to alanines (Supplementary Results, Supplementary Fig. 1a). Using NMR spectroscopy, we determined that wild-type (WT) p53TAD helicity (28%) increased to 64% when we replaced Pro27 with ...

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Section: Protein Purificationmentioning

confidence: 99%

“…2b). p53TAD and p53TAD P12A P13A bound to Mdm2 with similar affinities, whereas p53TAD P27A and p53TAD 3xA displayed approximately tenfold reductions in their dissociation constants [15][16][17] (Fig. 1c and Supplementary Fig.…”

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confidence: 99%

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

et al. 2014

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“…The up-regulation of miRNA-509-5p in HGC-27 cells could inhibit MDM2 expression, thus inhibiting cell proliferation, invasion, and migration. Some studies reported that MDM2 could work as a drug target for prostate cancer gene therapy, as the degradation of MDM2 could elevate intracellular p53 levels and potentiate p53 restrictive effects during the G1/S and G2/M phases, arresting cells at the G1/G2 phase and inducing apoptosis (Yin et al, 2003;Li et al, 2010). However, the exact underlying mechanism of regulation of MDM2 expression by miRNA-509-5p could not be elucidated and is the main limitation of the present study.…”

Section: Discussionmentioning

confidence: 80%

Inhibition of invasion and migration of prostate cancer cells by miRNA-509-5p via targeting MDM2

Tian

Luo

et al. 2017

Genet. Mol. Res.

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ABSTRACT. Prostate cancer is a common malignancy of the male reproductive-urinary system. MDM2 is an oncogene, whose expression can be regulated by microRNA (miRNA). The present study investigated the expression and correlation of miRNA-509-5p and MDM2 to determine the mechanism of their function in invasion and migration of prostate cancer cells. RT-PCR was performed to detect the expression of miRNA-509-5p and MDM2 in tumor, tumor-adjacent, and normal tissues, obtained from prostate cancer patients, using the HGC-27 cell line as an in vitro model. Cultured HGC-27 cells were transfected with miRNA-509-5p mimics, miRNA-509-5p inhibitor, and mimic control. Expression levels of miRNA-509-5p and MDM2 were quantified by RT-PCR. Cell proliferation and invasion/migration were examined by the MTT and transwell assays, respectively. MiRNA-509-5p was 2 X.M. Tian et al. Genetics and Molecular Research 16 (1): gmr16019195significantly down-regulated in prostate cancer cells exhibiting high MDM2 mRNA levels. MiRNA mimic transfection elevated miRNA levels and suppressed MDM2 expression. With prolonged incubation time, the proliferation ratio and OD values of miRNA-509-5p mimic transfected cells decreased, along with decrease in cell migration and invasion. These results suggested that miRNA-509-5p negatively regulates MDM2 expression via targeting the 3'-UTR of genes. As a novel tumor suppressor, miRNA-509-5p in prostate cancer HGC-27 cells can suppress MDM2 expression and inhibit cell proliferation, invasion, and migration. Therefore, miRNA-509-5p could be used as a novel therapeutic agent in the treatment of prostate cancer.

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“…1) reveals that there are 2 putative sites for phosphorylation that lie within the p53 binding region: Thr18 and Ser20. The suggestion that phosphorylation of these sites destabilizes p53-MDM2 interaction is supported by alanine mutagenesis experiments 23 and computer simulations. 18,24,39,40 The physical rationale for the destabilization of binding is usually ascribed to thermodynamic effects 18 however, kinetic effects have not been studied in either experimental or simulation studies.…”

Section: Resultsmentioning

confidence: 88%

“…Phosphorylation of Thr18 was shown to weaken p53 MDM2 binding by approximately one order of magnitude 19,22 and the Thr18Ala mutation was found to reduce the binding affinity of p53 to MDM2 by roughly a factor of 3. 23 Computational models, partly validated by experiments, suggested that the effect of phosphorylation was attributed to enhancing elecrostatic repulsion in the region where Thr18 docks by placing the negatively charged phosphorylated Thr18 near anionic regions of the MDM2 surface. 18,24 Phosphorylation of Ser20, however, was found to be sufficient to disrupt the p53-MDM2 interaction.…”

Section: Introductionmentioning

confidence: 98%

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

et al. 2015

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T he interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.

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

Cited by 124 publications

References 58 publications

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

Inhibition of invasion and migration of prostate cancer cells by miRNA-509-5p via targeting MDM2

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

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