Dataset Information

Nanosensing protein allostery using a bivalent mouse double minute two (MDM2) assay.

ABSTRACT: The tumor suppressor protein, p53, is either mutated or absent in >50% of cancers and is negatively regulated by the mouse double minute (MDM2) protein. Understanding and inhibition of the MDM2-p53 interaction are, therefore, critical for developing novel chemotherapeutics, which are currently limited because of a lack of appropriate study tools. We present a nanosensing approach to investigate full-length MDM2 interactions with p53, thus providing an allosteric assay for identifying binding ligands. Surface-enhanced Raman scattering (SERS)-active nanoparticles, functionalized with a p53 peptide mimic (peptide 12.1), display biologically specific aggregation following addition of MDM2. Nanoparticle assembly is competitively inhibited by the N-terminal MDM2-binding ligands peptide 12.1 and Nutlin-3. This study reports nanoparticle assembly through specific protein-peptide interactions that can be followed by SERS. We demonstrate solution-based MDM2 allosteric interaction studies that use the full-length protein.

SUBMITTER: Robson AF

PROVIDER: S-EPMC3361394 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nanosensing protein allostery using a bivalent mouse double minute two (MDM2) assay.

Robson Anna F AF Hupp Ted R TR Lickiss Fiona F Ball Kathryn L KL Faulds Karen K Graham Duncan D

Proceedings of the National Academy of Sciences of the United States of America 20120503 21

The tumor suppressor protein, p53, is either mutated or absent in >50% of cancers and is negatively regulated by the mouse double minute (MDM2) protein. Understanding and inhibition of the MDM2-p53 interaction are, therefore, critical for developing novel chemotherapeutics, which are currently limited because of a lack of appropriate study tools. We present a nanosensing approach to investigate full-length MDM2 interactions with p53, thus providing an allosteric assay for identifying binding lig ...[more]

PMID: 22556265

Similar Datasets

Project description:Deficiency of the tumour suppressor merlin leads to the development of schwannomas, meningiomas and ependymomas occurring spontaneously or as a part of the hereditary disease Neurofibromatosis type 2 (NF2). Merlin loss is also found in a proportion of other cancers like mesothelioma, melanoma, breast cancer and glioblastoma. The tumour suppressor/transcription factor p53 regulates proliferation, survival and differentiation and its deficiency plays a role in the development of many tumours. 53 can be negatively regulated by FAK, PI3K/AKT and MDM2 and possibly positively regulated by merlin in different cell lines. In this study we investigated the role of p53 in merlin-deficient tumours. Using our in vitro model of primary human schwannoma cells we have previously demonstrated that FAK is overexpressed/activated and localises into the nucleus of schwannoma cells increasing proliferation. AKT is strongly activated via platelet-derived growth factor (PDGF) - and insulin-like growth factor 1 (IGF1) - receptors increasing survival. Here we investigated p53 regulation and its role in proliferation and survival of human primary schwannoma cells using western blotting, immunocytochemistry, immunohistochemistry and proliferation, survival and transcription factor assays. In human primary schwannoma cells p53 was found to be downregulated while MDM2 was upregulated leading to increased cell proliferation and survival. p53 is regulated by merlin involving FAK, AKT and MDM2. Merlin reintroduction into schwannoma cells increased p53 levels and activity, and treatment with Nutlin-3, a drug which increases p53 stability by disrupting the p53/MDM2 complex, decreased tumour growth and reduced cell survival. These findings are important to dissect the mechanisms responsible for the development of merlin-deficient tumours and to identify new therapeutic targets. We suggest that Nutlin-3, possibly in combination with FAK or PI3K inhibitors, can be employed as a novel treatment for schwannoma and other merlin-deficient tumours.

Project description:The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

Dataset Information

Nanosensing protein allostery using a bivalent mouse double minute two (MDM2) assay.

Publications

Nanosensing protein allostery using a bivalent mouse double minute two (MDM2) assay.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets