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Dra.
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
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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
SUMMARY
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.
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