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Publication Abstract from PubMed

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERalpha), and small molecule stabilization of the ERalpha/14-3-3zeta protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERalpha-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERalpha; however, structural and mechanistic insights into ERalpha/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERalpha/14-3-3zeta complex by isolating 14-3-3zeta in complex with an ERalpha protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERalpha/14-3-3zeta complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERalpha homodimer and the 14-3-3zeta homodimer. 14-3-3zeta binding to ERalpha, and ERalpha/14-3-3zeta complex stabilization by FC-A, appeared to be orthogonal to ERalpha endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERalpha antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERalpha LBD while ERalpha was bound to 14-3-3zeta. Furthermore, stabilization of the ERalpha/14-3-3zeta protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERalpha-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERalpha via the ERalpha/14-3-3 complex as an alternative drug discovery approach.

Molecular basis and dual ligand regulation of tetrameric estrogen receptor alpha/14-3-3zeta protein complex.,Somsen BA, Sijbesma E, Leysen S, Honzejkova K, Visser EJ, Cossar PJ, Obsil T, Brunsveld L, Ottmann C J Biol Chem. 2023 Jul;299(7):104855. doi: 10.1016/j.jbc.2023.104855. Epub 2023 , May 22. PMID:37224961^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.