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Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

ABSTRACT: Estrogen receptor alpha (ER?) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ER?, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ER? dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ER? dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ER?/14-3-3 interaction. Cocrystallization of the trimeric ER?/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ER?-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ER? phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T(594)-specific antibody and by mass spectrometry. In line with its ER?/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ER? dimerization, inhibits ER?/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ER? are provided, together with a small molecule that selectively targets this ER?/14-3-3 interface.

SUBMITTER: De Vries-van Leeuwen IJ

PROVIDER: S-EPMC3670367 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

De Vries-van Leeuwen Ingrid J IJ da Costa Pereira Daniel D Flach Koen D KD Piersma Sander R SR Haase Christian C Bier David D Yalcin Zeliha Z Michalides Rob R Feenstra K Anton KA Jiménez Connie R CR de Greef Tom F A TF Brunsveld Luc L Ottmann Christian C Zwart Wilbert W de Boer Albertus H AH

Proceedings of the National Academy of Sciences of the United States of America 20130515 22

Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers ...[more]

PMID: 23676274

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Project description:BackgroundMost proteins function by interacting with other molecules. Their interaction interfaces are highly conserved throughout evolution to avoid undesirable interactions that lead to fatal disorders in cells. Rational drug discovery includes computational methods to identify the interaction sites of lead compounds to the target molecules. Identifying and classifying protein interaction interfaces on a large scale can help researchers discover drug targets more efficiently.DescriptionWe introduce a large-scale protein domain interaction interface database called InterPare http://interpare.net. It contains both inter-chain (between chains) interfaces and intra-chain (within chain) interfaces. InterPare uses three methods to detect interfaces: 1) the geometric distance method for checking the distance between atoms that belong to different domains, 2) Accessible Surface Area (ASA), a method for detecting the buried region of a protein that is detached from a solvent when forming multimers or complexes, and 3) the Voronoi diagram, a computational geometry method that uses a mathematical definition of interface regions. InterPare includes visualization tools to display protein interior, surface, and interaction interfaces. It also provides statistics such as the amino acid propensities of queried protein according to its interior, surface, and interface region. The atom coordinates that belong to interface, surface, and interior regions can be downloaded from the website.ConclusionInterPare is an open and public database server for protein interaction interface information. It contains the large-scale interface data for proteins whose 3D-structures are known. As of November 2004, there were 10,583 (Geometric distance), 10,431 (ASA), and 11,010 (Voronoi diagram) entries in the Protein Data Bank (PDB) containing interfaces, according to the above three methods. In the case of the geometric distance method, there are 31,620 inter-chain domain-domain interaction interfaces and 12,758 intra-chain domain-domain interfaces.

Project description:BackgroundThe 14-3-3 proteins are structurally conserved throughout eukaryotes and participate in protein kinase signaling. All 14-3-3 proteins are known to bind to evolutionally conserved phosphoserine-containing motifs (modes 1 and/or 2) with high affinity. In Trypanosoma brucei, 14-3-3I and II play pivotal roles in motility, cytokinesis and the cell cycle. However, none of the T. brucei 14-3-3 binding proteins have previously been documented.Methodology/principal findingsInitially we showed that T. brucei 14-3-3 proteins exhibit far lower affinity to those peptides containing RSxpSxP (mode 1) and RxY/FxpSxP (mode 2) (where x is any amino acid residue and pS is phosphoserine) than human 14-3-3 proteins, demonstrating the atypical target recognition by T. brucei 14-3-3 proteins. We found that the putative T. brucei protein phosphatase 2C (PP2c) binds to T. brucei 14-3-3 proteins utilizing its mode 3 motif (-pS/pTx(1-2)-COOH, where x is not Pro). We constructed eight chimeric PP2c proteins replacing its authentic mode 3 motif with potential mode 3 sequences found in Trypanosoma brucei genome database, and tested their binding. As a result, T. brucei 14-3-3 proteins interacted with three out of eight chimeric proteins including two with high affinity. Importantly, T. brucei 14-3-3 proteins co-immunoprecipitated with an uncharacterized full-length protein containing identified high-affinity mode 3 motif, suggesting that both proteins form a complex in vivo. In addition, a synthetic peptide derived from this mode 3 motif binds to T. brucei 14-3-3 proteins with high affinity.Conclusion/significanceBecause of the atypical target recognition of T. brucei 14-3-3 proteins, no 14-3-3-binding proteins have been successfully identified in T. brucei until now whereas over 200 human 14-3-3-binding proteins have been identified. This report describes the first discovery of the T. brucei 14-3-3-binding proteins and their binding motifs. The high-affinity phosphopeptide will be a powerful tool to identify novel T. brucei 14-3-3-binding proteins.

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Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

Publications

Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

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