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Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

ABSTRACT: 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 (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

SUBMITTER: Somsen BA

PROVIDER: S-EPMC10302166 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

Somsen Bente A BA Sijbesma Eline E Leysen Seppe S Honzejkova Karolina K Visser Emira J EJ Cossar Peter J PJ Obšil Tomáš T Brunsveld Luc L Ottmann Christian C

The Journal of biological chemistry 20230522 7

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 (ERα), and small molecul ...[more]

PMID: 37224961

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Project description:ObjectiveMetabolic stress primes monocytes for accelerated chemokine-mediated adhesion, migration, and recruitment into vascular lesions by increasing actin remodeling. The mechanism linking metabolic stress to accelerated actin turnover and enhanced monocyte migration was not known. We tested the hypothesis that in metabolically primed monocytes, the acceleration of monocyte chemoattractant protein-1-induced chemotaxis is mediated by the hyperactivation of cofilin.Approach and resultsMetabolic priming was induced by exposing human THP-1 monocytes to diabetic conditions, that is, human native low-density lipoprotein plus high glucose concentrations. In healthy monocytes, monocyte chemoattractant protein-1 induced the phosphorylation and inactivation of cofilin. This response was completely blocked in metabolically primed monocytes but restored by overexpression of the thiol transferase, glutaredoxin 1. Cofilin kinase, LIM kinase 1, and cofilin phosphatase, Slingshot-1L, were not affected by metabolic stress. However, metabolic priming increased 3.8-fold the S-glutathionylation of the Slingshot-1L-binding protein 14-3-3ζ (zeta), resulting in its caspase-dependent degradation. Glutaredoxin 1 overexpression inhibited low-density lipoprotein plus high glucose-induced S-glutathionylation and degradation of 14-3-3ζ. The C25S mutant of 14-3-3ζ was resistant to both S-glutathionylation and degradation induced by low-density lipoprotein plus high glucose. Overexpression of the C25S mutant restored monocyte chemoattractant protein-1-induced cofilin phosphorylation and prevented accelerated migration of metabolically stressed monocytes, suggesting that loss of 14-3-3ζ increases the pool of free Slingshot-1L phosphatase, thereby preventing the phosphorylation and deactivation of cofilin in response to chemokine activation.ConclusionsBy preventing the inactivation of cofilin, metabolic stress-induced degradation of 14-3-3ζ promotes the conversion of blood monocytes into a hypermigratory, proatherogenic phenotype.

Dataset Information

Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

Publications

Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

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