Maria Izabel Chiamolera
THE PROLINE IN CODON 452 OF THYROID RECEPTOR b (TR b ) PLAYS A CRITICAL ROLE IN THE INTERACTION WITH COREPRESSOR AND COACTIVATOR MARIA IZABEL CHIAMOLERA, RUTNÉIA P. PESSANHA, GUSTAVO B. BARRA, LUCAS BLEICHER, IGOR POLIKARPOV, FRANCISCO A. R. NEVES, RUI M. B. MACIEL
Federal University of São Paulo (MIC, RMBM); University of Brasília (RPP, GBB, FARN); Physics Institute of São Carlos, University of São Paulo (LB, IP); Brazil
All mutations of Thyroid Receptor b (TRb) observed in Resistance to Thyroid Hormone (RTH) are in the receptor’s ligand-binding domain (LBD). The 12 helixes of this domain formed an hydrophobic pocket where the ligand is deeply buried. Upon ligand binding, the carboxy-terminal (Helix 12) undergoes major conformational changes, from a more open conformation to a closed one, resulting in dissociation of corepressor and formation of a surface that may determine whether coactivators can interact with TR, an essential requirement for TR transactivation function.
In the present study, we report and characterize a novel mutation in a patient with RTH, P452L, using conventional molecular methods including T3-binding assay, transient reporter gene transfection analysis in positive and negative regulated gene, glutathione-S-transferase (GST) pull-down assay, EMSA and homology modeling of the protein.
At the luciferase assays, P452L exhibited a significant transcriptional impairment in both positive (DR4 and F2) and negative regulated genes (TRH) and did not recovered its transcriptional activity, even at supramaximal doses of T3, as other mutant (A317V) despite the fact that T3 binding in both mutants was similar. The gel shift with in vitro produced labeled proteins, performed in DR4 and F2 response elements, showed simila bind to DNA and formation of complexes betwen P452L mutant and wild type.In GST pull down assay, P452L failed to completely release the corepressor binding and exhibited almost no binding to the coactivator.
Because our results demonstrated that this mutant was disrupting coactivator interaction we decided to study better our protein structure using Computer modelling by homology with hTRb complexed with GRIP1, more than 100 models were constructed with the Modeller progam, and validated using specific softwares, but unfortunetaly, at computer model we could not demonstraded great changes in TR structure. So crystal structure will be important to answer these structural questions.
In conclusion, similar to P452L, the 453 and 454 mutants failed to release the co-repressor and was unable to interact with co-activator in the presence of T3. The lack of T3 response in modulation of P452L transcriptional activity could not be attributed only to its reduced T3 binding affinity, but also to a disruption in the interaction with co-regulators Otherwise, the failure in repression of negative regulated genes could be explained by the abolished interaction with the coactivators as it was recently described by Ortiga-Carvalho et al. Therefore, we suggest that prolines 452 and 453 could cause the same effect in TR structure, disturbing the interactions with both coactivator and corepressor not only by alteration in T 3 binding, but mostly because both prolines may be responsible for a hinge function of helix 12, allowing its movement into the body of the LBD, inducing corepressor release and the formation of a coactivator interaction site.