Binding of Natively Unfolded HIF-1α ODD Domain to p53

Sánchez-Puig, Nuria; Veprintsev, Dmitry B.; Fersht, Alan R.

doi:10.1016/j.molcel.2004.11.019

Cited by 98 publications

(84 citation statements)

References 49 publications

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“…p53N (amino acids 1-93) cDNA was cloned into a modified pRSETa vector encoding an N-terminal 6ϫ His tag, lipoyl domain, and thrombin cleavage site to make the plasmid pHLT1-93. The expression and purification method were performed as described (36). 1-296).…”

Section: Purification Of Human Hdm2 N-terminal (Residues 2-125) Domainmentioning

confidence: 99%

The central region of HDM2 provides a second binding site for p53

Rüdiger

Veprintsev

et al. 2006

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

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132

141

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HDM2 is a negative regulator of p53 that inhibits its transcriptional activity and subjects it to degradation by an E3 ligase activity. The primary binding site for HDM2 on p53 is located in its N-terminal domain. A second site on the p53 core domain (p53C) binds to an unidentified site in HDM2. We found that this site is in its acidic domain and part of the zinc finger domain by examining the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. NMR spectroscopy showed that peptides derived from this region of HDM2 bound to residues in the specific DNA-binding site of p53C. The peptides were displaced from the site by gadd45 sequencespecific DNA. Phosphorylation of single amino acids in the central domain of HDM2 did not abolish the interaction between the HDM2-derived peptides and p53C. We speculate that this second binding site helps in stabilizing the interaction between HDM2 and p53 during p53 degradation.isothermal titration calorimetry ͉ Mdm2 ͉ NMR T he tumor suppressor protein p53 is important in maintaining genome stability and in preventing cancer development (1, 2). In response to various stress signals, p53 mediates cell-cycle arrest and apoptosis (3). p53 is a homotetramer consisting of an N-terminal transactivation domain, proline-rich regulatory domain, DNA-binding core domain (p53C), tetramerization domain, and C-terminal negative regulatory domain. Its major regulator, HDM2, is induced by p53 and acts as a feedback inhibitor (4). HDM2 regulates the activity of p53 in at least three ways. First, the N-terminal domain of HDM2 binds directly to p53's transactivation domain and inhibits its transcriptional function (5). Second, HDM2 acts as a ubiquitin ligase, targeting p53 and promoting its degradation (6, 7). Third, upon binding, HDM2 exports p53 from the nucleus to the cytoplasm (8).The interaction between peptides derived from the p53 N terminus and the HDM2-N-terminal domain has been extensively studied (9-12), and several compounds have been proposed to abolish this interaction (13-16). Recently, a new HDM2-binding site has been reported in p53C that plays a regulatory role in modulating p53 ubiquitination (17), although the exact binding site on HDM2 involved in this interaction has not yet been identified. There are conflicting speculations on its location at either the HDM2 N terminus (18) or in the acidic domain (19).Here, we examined the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. We found that the HDM2 N-terminal domain interacts only with p53's N-terminal domain, whereas from the peptide-array screen, we identified a second binding site for p53C located in the central domain (residues 221-302) of HDM2. We measured the binding affinity of the peptides to p53C by analytical ultracentrifugation. We identified the binding site on p53C by NMR and fluorescence anisotropy to be its DNA-binding site....

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Section: Purification Of Human Hdm2 N-terminal (Residues 2-125) Domainmentioning

confidence: 99%

The central region of HDM2 provides a second binding site for p53

Rüdiger

Veprintsev

et al. 2006

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

Self Cite

132

141

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“…HIF1␣ appears to enhance p53 activation by ␥-radiation (IR), resulting in increased p53 phosphorylation and p53-induced apoptosis (13). Furthermore, HIF1␣ has been shown to complex with p53 and promote its stability (14,15). In contrast, a role for HIF2␣ in p53 pathway regulation and cellular responses to IR has not been reported.…”

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confidence: 99%

HIF2α inhibition promotes p53 pathway activity, tumor cell death, and radiation responses

Bertout

Majmundar

Gordan

et al. 2009

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

177

163

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Approximately 50% of cancer patients receive radiation treatment, either alone or in combination with other therapies. Tumor hypoxia has long been associated with resistance to radiation therapy. Moreover, the expression of hypoxia inducible factors HIF1␣ and/or HIF2␣ correlates with poor prognosis in many tumors. Recent evidence indicates that HIF1␣ expression can enhance radiation-induced apoptosis in cancer cells. We demonstrate here that HIF2␣ inhibition promotes tumor cell death and, in contrast to HIF1␣, enhances the response to radiation treatment. Specifically, inhibiting HIF2␣ expression augments p53 activity, increases apoptosis, and reduces clonogenic survival of irradiated and non-irradiated cells. Moreover, HIF2␣ inhibition promotes p53-mediated responses by disrupting cellular redox homeostasis, thereby permitting reactive oxygen species (ROS) accumulation and DNA damage. These results correlate with altered p53 phosphorylation and target gene expression in untreated human tumor samples and show that HIF2␣ likely contributes to tumor cell survival including during radiation therapy.M any cellular responses to hypoxia are mediated by the hypoxia inducible factors (HIFs). These transcription factors promote the expression of over 200 genes (1) and are heterodimers consisting of either HIF1␣ or HIF2␣ bound to the HIF␤/ARNT subunit. While ARNT is constitutively expressed, both HIF␣ subunits are regulated by O 2 availability. Under normoxia, the von Hippel-Lindau (VHL) E3 ligase complex targets HIF␣ subunits for proteasomal degradation (2). When O 2 levels decline, the HIF␣ subunits are stabilized, bind ARNT, and activate the expression of target genes providing hypoxic adaptations.Solid tumors are characterized by oncogenic signaling and hypoxic microenvironments that promote HIF␣ accumulation (3). Moreover, HIF1␣ and/or HIF2␣ expression has been associated with increased tumor vascularization and poor prognosis of numerous cancers such as breast, ovarian, and non-small cell lung cancer (2, 4). Of note, in mouse xenograft models, HIF2␣ (and not HIF1␣) expression is crucial for growth of clear cell renal cell carcinoma (ccRCC) (5, 6) and neuroblastoma (7) tumors.TP53 is a tumor suppressor that is mutated or silenced in a majority of human cancers (8). It coordinates many cellular stress responses by regulating genes involved in DNA repair, cell cycle arrest, and apoptosis. Following stress stimuli, p53 is activated through a variety of post-translational modifications, including phosphorylation on serine 15 (9). For example, the ataxia telangiectasia mutated (ATM) and checkpoint kinase 2 (Chk2) kinases directly phosphorylate p53 in response to DNA damage, resulting in its activation (9). Although many tumors select for TP53 mutations, p53 pathway inhibition can also contribute to tumor progression (10).With the emergence of HIF inhibitors (11,12) and their potential use in cancer therapy, it is important to accurately predict the response of HIF␣-expressing tumor cells to treatment. HIF1␣ appears to enh...

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“…The concentrationdependent increase of RU signals clearly confirms that there are specific interactions between the Fab and all the epitope-near preS1 regions. The interactions of these four preS1 peptides for the Fab were weak with the equilibrium dissociation constant (K d ) values of 1-2 mM range: 1.54, 1.63, 2.04, and 1.99 mM for preS1 (1-11), preS1 (27)(28)(29)(30)(31)(32)(33)(34)(35), preS1 (66-76), and preS1 (95-104), respectively. Although this binding affinity seems low, it is comparable to the affinities observed for germline antibody binding [28] and TCR-peptide/MHC (pMHC) interactions [35].…”

Section: Resultsmentioning

confidence: 99%

“…HzKR127 Fab in 25 mM sodium acetate, pH 5.5 was immobilized to a channel of CM5 sensor chip using an amine coupling kit (BIAcore AB) and the control flow-subtracted sensograms are obtained. The peptide concentrations range from 10 to 640 nM for preS1 (36-46), from 5 to 80 nM for preS1 (1-119), and from 100 to 800 lM for preS1 (1-11), preS1 (27)(28)(29)(30)(31)(32)(33)(34)(35), preS1 (66-76), and preS1 (95-104). Kinetic measurements were made and kinetic constants were derived with the BIAevaluation Version 3.0 software (BIAcore AB).…”

Section: Surface Plasma Resonance Experimentsmentioning

confidence: 99%

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Broadly neutralizing anti‐HBV antibody binds to non‐epitope regions of preS1

Chi

Kim

et al. 2009

FEBS Letters

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a b s t r a c tBroadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.

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Binding of Natively Unfolded HIF-1α ODD Domain to p53

Cited by 98 publications

References 49 publications

The central region of HDM2 provides a second binding site for p53

The central region of HDM2 provides a second binding site for p53

HIF2α inhibition promotes p53 pathway activity, tumor cell death, and radiation responses

Broadly neutralizing anti‐HBV antibody binds to non‐epitope regions of preS1

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