Transcriptional regulation controlled by thyroid hormone receptor (TR) drives events such as development, differentiation, and metabolism. TRs may act either as homodimers or as heterodimers with retinoid X receptor (RXR). Thyroid hormone T3 preferentially binds TR-RXR heterodimers, which activate transcription through coactivator recruitment. However, it is unclear whether TR-RXR heterodimers may also be responsive to the canonical RXR agonist 9-cis retinoic acid (9C) in the context of physiological gene regulation. New structural studies suggest that 9C promotes the displacement of bound coactivators from the heterodimer, modifying TR-RXR activity. To shed light on the molecular mechanisms that control TR-RXR function, we used biophysical approaches to characterize coregulator recruitment to TR-TR or to TR-RXR in the presence of T3 and/or 9C as well as cell-based assays to establish the functional significance of biophysical findings. Using cell-based and fluorescence assays with mutant and wild-type TR, we show that 9C does indeed have a function in the TR-RXR heterodimer context, in which it induces the release of corepressors. Furthermore, we show that 9C does not promote detectable conformational changes in the structure of the TR-RXR heterodimer and does not affect coactivator recruitment. Finally, our data support the view that DNA binding domain and Hinge regions are important to set up NR-coactivator binding interfaces. In summary, we showed that the RXR agonist 9C can regulate TR function through its modulation of corepressor dissociation.

Thyroid hormone receptors (TRs) are responsible for mediating thyroid hormone (T3 and T4) actions at a cellular level. They belong to the nuclear receptor (NR) superfamily and execute their main functions inside the cell nuclei as hormone-regulated transcription factors. These receptors also exhibit so-called “non-classic” actions, for which other cellular proteins, apart from coregulators inside nuclei, regulate their activity. Aiming to find alternative pathways of TR modulation, we searched for interacting proteins and found that PDIA1 interacts with TRβ in a yeast two-hybrid screening assay. The functional implications of PDIA1—TR interactions are still unclear; however, our co-immunoprecipitation (co-IP) and fluorescence assay results showed that PDI was able to bind both TR isoforms in vitro. Moreover, T3 appears to have no important role in these interactions in cellular assays, where PDIA1 was able to regulate transcription of TRα and TRβ-mediated genes in different ways depending on the promoter region and on the TR isoform involved. Although PDIA1 appears to act as a coregulator, it binds to a TR surface that does not interfere with coactivator binding. However, the TR:PDIA1 complex affinity and activation are different depending on the TR isoform. Such differences may reflect the structural organization of the PDIA1:TR complex, as shown by models depicting an interaction interface with exposed cysteines from both proteins, suggesting that PDIA1 might modulate TR by its thiol reductase/isomerase activity.

Estrogen receptor α (ERα) is a regulatory protein that can access a set of distinct structural configurations. ERα undergoes extensive remodeling as it interacts with different agonists and antagonists, as well as transcription activation and repression factors. Moreover, breast cancer tumors resistant to hormone therapy have been associated with the imbalance between the active and inactive ERα states. Cancer-activating mutations in ERα play a crucial role in this imbalance and can promote the progression of cancer. However, the rate of this progression can also be increased by dysregulated pH in the tumor microenvironment. Many molecular aspects of the process of activation of ERα that can be affected by these pH changes and mutations are still unclear. Thus, we applied computational and experimental techniques to explore the activation process dynamics of ER for environments with different pHs and in the presence of one of the most recurrent cancer-activating mutations, D538G. Our results indicated that the effect of the pH increase associated with the D538G mutation promoted a robust stabilization of the active state of ER. We were also able to determine the main protein regions that have the most potential to influence the activation process under different pH conditions, which may provide targets of future therapeutics for the treatment of hormone-resistant breast cancer tumors. Finally, the approach used here can be applied for proteins associated with the proliferation of other cancer types, which can also have their function affected by small pH changes.