p53 Promotes proteasome-dependent degradation of oncogenic protein HBx by transcription of MDM2

Hepatitis B virus X protein (HBx) is closely involved in the development of hepatocellular carcinoma (HCC). Tumor suppressor p53 was reported to induce HBx degradation and repress its oncogenic function recently, but the molecular mechanism is unknown. In this study, we attempted to identify the underlying mechanism. We found that overexpression of p53 protein reduces the level of HBx protein and shortens its half-life, however, in MDM2 knock out cells, p53 has no effects on degradation of HBx, meanwhile, overexpression of MDM2 in absence of p53 can accelerate turnover of HBx protein. These indicate that p53-mediated HBx degradation is MDM2-dependent. MDM2 interacts with HBx in vitro and in vivo but does not promote its ubiquitination. In consistent with the results above, HCC tissue samples with wild-type p53 hardly detect HBx protein, whereas, HBx always accumulate in the tissues with mutant p53. Our data provide a possible mechanism on how p53 regulate HBx stability and also a new clue for the study of p53 mutation and HCC development.     

Binding of p53-derived ligands to MDM2 induces a variety of long range conformational changes

We have used NMR to study the effects of peptide binding on the N-terminal p53-binding domain of human MDM2 (residues 25-109). There were changes in HSQC-chemical shifts throughout the domain on binding four different p53-derived peptide ligands that were significantly large to be indicative of global conformational changes. Large changes in chemical shift were observed in two main regions: the peptide-binding cleft that directly binds the p53 ligands; and the hinge regions connecting the beta-sheet and alpha-helical structures that form the binding cleft. These conformational changes reflect the adaptation of the cleft on binding peptide ligands that differ in length and amino acid composition. Different ligands may induce different conformational transitions in MDM2 that could be responsible for its function. The dynamic nature of MDM2 might be important in the design of anti-cancer drugs that are targeted to its p53-binding site.     

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.     

BMRP is a Bcl-2 binding protein that induces apoptosis

Members of the Bcl-2 family of proteins play important roles in the regulation of cell death by apoptosis. The yeast Two-Hybrid system was utilized to identify a protein that interacts with the anti-apoptotic protein Bcl-2, designated BMRP. This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41). Binding experiments confirmed the interaction of BMRP to Bcl-2 in mammalian cells. Subcellular fractionation by differential centrifugation studies showed that both Bcl-2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria). Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues. Additionally, a larger 2.2 kb mRNA species was also observed in some tissues. Western blot analysis showed that endogenous BMRP runs as a band of 16-17 kDa in SDS-PAGE. Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells. Transfection of BMRP showed similar effects to those observed by overexpression of the pro-apoptotic proteins Bax or Bad. BMRP-stimulated cell death was counteracted by co-expression of Bcl-2. The baculoviral caspase inhibitor p35 also protected cells from BMRP-induced cell death. These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl-2 and caspases.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53

The p53 tumour suppressor protein is tightly controlled by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), but maintains MDM2 expression as part of a negative feedback loop. We have identified the immunophilin, 25 kDa FK506-binding protein (FKBP25), previously shown to be regulated by p53-mediated repression, as an MDM2-interacting partner. We show that FKBP25 stimulates auto-ubiquitylation and proteasomal degradation of MDM2, leading to the induction of p53. Depletion of FKBP25 by siRNA leads to increased levels of MDM2 and a corresponding reduction in p53 and p21 levels. These data are consistent with the idea that FKBP25 contributes to regulation of the p53-MDM2 negative feedback loop.

Structured summary

MINT-6823686:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by anti bait coimmunoprecipitation (MI:0006)MINT-6823707, MINT-6823722:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q62446) by pull down (MI:0096)MINT-6823775:P53 (uniprotkb:Q04637) physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by anti bait coimmunoprecipitation (MI:0006)MINT-6823735, MINT-6823749:FKBP25 (uniprotkb:Q62446) binds (MI:0407) to MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823761:Ubiquitin (UNIPROTKB:62988)P physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823669:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by two hybrid (MI:0018)     

Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation

The tumor suppressor p53 is stabilized and activated in response to cellular stress through post-translational modifications including acetylation. p300/CBP-mediated acetylation of p53 is negatively regulated by MDM2. Here we show that MDM2 can promote p53 deacetylation by recruiting a complex containing HDAC1. The HDAC1 complex binds MDM2 in a p53-independent manner and deacetylates p53 at all known acetylated lysines in vivo. Ectopic expression of a dominant-negative HDAC1 mutant restores p53 acetylation in the presence of MDM2, whereas wild-type HDAC1 and MDM2 deacetylate p53 synergistically. Fibroblasts overexpressing a dominant negative HDAC1 mutant display enhanced DNA damage-induced p53 acetylation, increased levels of p53 and a more pronounced induction of p21 and MDM2. These results indicate that acetylation promotes p53 stability and function. As the acetylated p53 lysine residues overlap with those that are ubiquitylated, our results suggest that one major function of p53 acetylation is to promote p53 stability by preventing MDM2-dependent ubiquitylation, while recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.     

Inhibition of MDM2 by hsp90 contributes to mutant p53 stabilization

Stabilization and overexpression are hallmarks of mutant p53 found in nearly 50% of human tumors. Mutations in the conformation-sensitive core domain of p53 often lead to association with molecular chaperones such as hsp70 and hsp90. Inhibition of hsp90 function accelerates mutant p53 degradation. We recently found that expression of p53 core domain mutants inhibits MDM2 degradation, suggesting that mutant p53 can modulate MDM2 functions. In this report, we show that mutant p53 mediates formation of MDM2-p53-hsp90 complexes. Release of MDM2 from the p53-hsp90 complex after DNA damage restores MDM2 but not p53 turnover, whereas dissociation of hsp90 by geldanamycin increases the degradation of both MDM2 and mutant p53. Mutant p53 degradation after hsp90 inhibition requires MDM2 expression. The interaction between MDM2 and hsp90 is disrupted by the 2A10 antibody, which recognizes a site on MDM2 important for binding to alternative reading frame (ARF). Expression of mutant p53 prevents MDM2 from binding ARF and accumulating in the nucleolus in an hsp90-dependent fashion. These results suggest that hsp90 recruited by mutant p53 conceals the ARF-binding site on MDM2 and inhibits its ubiquitin-protein isopeptide ligase function, resulting in the stabilization of both mutant p53 and MDM2.     

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.