Structure-based Design of High Affinity Peptides Inhibiting the Interaction of p53 with MDM2 and MDMX

Phan, Jason; Li, Zhenyu; Kasprzak, Agnieszka; Li, Baozong; Sebti, Saı̈d M.; Guida, Wayne C.; Schönbrunn, E.; Chen, Jiandong

doi:10.1074/jbc.m109.073056

Cited by 137 publications

(170 citation statements)

References 44 publications

(92 reference statements)

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“…Atomic structures have been obtained for the MDMX N-terminal domain and RING domain in isolation (24,25). Our results suggest that in the context of the full-length protein, these domains are often present in complex with the central domain.…”

Section: Discussionmentioning

confidence: 94%

Autoinhibition of MDMX by intramolecular p53 mimicry

Chen¹,

Borcherds

Wu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.T he p53 tumor suppressor is activated by numerous cellular and environmental signals and induces the expression of genes that regulate metabolism, cell growth, cell cycle, apoptosis, and senescence (1). MDM2 and MDMX are key regulators that control the cellular level and transcriptional activity of p53 through direct binding. Mouse knockout experiments showed that both MDM2 and MDMX are essential for controlling p53 activity during embryogenesis. Somatic knockout experiments showed that MDM2 is indispensable for regulating p53 in adult tissues, whereas MDMX deletion does not lead to cell death (2). MDM2 is a well-established p53 transcriptional target that forms a negative feedback loop by binding to the N-terminal transcriptional activation domain of p53 and, subsequently, ubiquitinating the C-terminal regulatory domain, which leads to degradation of p53 by the proteasome. p53 binding sites are also found in intron 1 of human MDMX, and p53 activation leads to moderate induction of MDMX transcription (3). Therefore, MDMX is a p53 target gene that may also provide dynamic feedback in response to p53 activation.MDMX alone does not have E3 ligase activity, but it is important for regulating p53 transcriptional function. MDMX expression and phosphorylation by the ATM/Chk2 pathway is important for the p53-mediated DNA damage response in mice (4, 5). MDMX levels are controlled by MDM2-mediated ubiquitination in a stress-dependent fashion (6, 7). Significant degradation of MDMX occurs after DNA damage through phosphorylation at several C-terminal sites (S342 and S367 by Chk2, S403 by ATM) (8). Furthermore, ribosomal stress promotes MDMX degradation through L11-MDM2 interaction (9), and oncogenic stress promotes MDMX degradation through ARF expression (10). Therefore, key signaling mechanisms that block p53 degradation simultaneously enhance MDMX degradation by MDM2. These observations underscore the co...

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Section: Discussionmentioning

confidence: 94%

Autoinhibition of MDMX by intramolecular p53 mimicry

Chen¹,

Borcherds

Wu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The authors' analyses determine which p53 residues are important for binding to the hydrophobic pocket. In addition to the classic residues 16,17 (F19, L22, W23 and L26), favorable electrostatic contributions from N29 can form a salt bridge with E25 of MDM2 (Fig. 2B) accounting partly for the increased affinity for MDM2 over MDMX.…”

Section: It Is Not Just Mdm2: Other Dynamic Peptide-protein Interactimentioning

confidence: 99%

The molecular dynamics of MDM2

Nicholson¹,

Hupp²

2010

Cell Cycle

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“…Using the information from our recent study (33), seven amino acid substitutions were introduced into p53 N terminus (33). The resulting full-length DI-p53 showed increased binding to MDM2 (ϳ6-fold) in coimmunoprecipitation assay (Fig.…”

Section: Mdm2 Ring Domain Interaction and Regulation In Vivomentioning

confidence: 99%

Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

Qian

Cross

et al. 2011

Molecular and Cellular Biology

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MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.The p53 pathway is critical for maintenance of genomic stability and preventing development of cancer. The most notable feature of p53 is its stabilization after exposure to a wide range of stress signals such as hyperproliferation, nucleotide depletion, and DNA damage. These responses may be essential for its function as a tumor suppressor (14). In normal cells, p53 is present at very low levels due to rapid degradation through the ubiquitin-dependent proteasome pathway. p53 turnover is regulated by MDM2, which binds p53 and functions as an ubiquitin E3 ligase to promote p53 degradation by the proteasome (15,16,21). Additional E3 ligases such as Pirh2 and Cop1 have also been implicated as regulators of p53 turn over (12,24). However, their physiological significance in p53 regulation and stress response still remain to be established. Current evidence suggests that MDM2 is a major and indispensable regulator of p53 level (18,32).MDM2 and p53 interact through their N-terminal domains in a high-affinity binding and through their central domains in a weak binding (48). Upon complex formation, the MDM2 C-terminal RING domain recruits ubiquitin-conjugating enzyme E2 that performs the transfer of activated ubiquitin to p53 lysine residues. To date, the understanding of molecular mechanisms that lead to p53 stabilization by different pathways remain incomplete. An important group of MDM2 regulators are proteins that bind to the central acidic domain, such as ARF, L5, L11, and L23 (37, 50). These basic proteins are important for mediating mitogenic stress and ribosomal stress signals to p53. They have been shown to inhibit MDM2-mediated p53 ubiquitination, but little is known about the mechanisms. They may act by neutralizing ...

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Structure-based Design of High Affinity Peptides Inhibiting the Interaction of p53 with MDM2 and MDMX

Cited by 137 publications

References 44 publications

Autoinhibition of MDMX by intramolecular p53 mimicry

Autoinhibition of MDMX by intramolecular p53 mimicry

The molecular dynamics of MDM2

Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

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