Investigating The Role Of Acetylation In The Nucleocytoplasmic Shuttling Of Trα1 Wild-Type And The Resistance To Thyroid Hormone Syndrome C392X Mutant

The thyroid hormone receptor α 1 (TRα1) is one of three functional subtypes of the thyroid hormone receptor (TR). In general, TRs are nuclear receptors, ligand-binding transcription factors that interact with thyroid hormone to regulate growth, development and metabolism. Post-translational modifications, like acetylation, impact protein structure and diversify protein function. Post-translational acetylation of TRα1 occurs at lysine residues located within its nuclear localization signal 1 (NLS-1). Dynamic nucleocytoplasmic shuttling is also a characteristic of TRα1, and the cause and significance of this is of great interest. Based on the location of the acetylation sites of TRα1 within NLS-1, it seemed reasonable to investigate the effect of acetylation on the receptor’s nucleocytoplasmic shuttling. Previously published work showed that mCherry-tagged TRα1 acetylation mimics had increased cytoplasmic localization and enhanced gene transactivation, while the mCherry-tagged TRα1 nonacetylation mimics had increased nuclear retention and impaired gene transactivation. The subcellular localization of these mimics supports that acetylation plays a role in the cellular translocation of TRα1. In the current study, we performed immunoprecipitation assays, using acetyl lysine affinity beads, to investigate the effect of actual acetylation status on TRα1 nucleocytoplasmic shuttling, and hypothesized that acetylation does increase the cytoplasmic localization of TRα1. Additionally, the C392X mutation in TRα1 is implicated in Resistance to Thyroid Hormone syndrome α (RTH-α), a disease characterized by mutant receptors that have reduced binding affinity for thyroid hormone, which negatively affects metabolism. In this study, we also investigated the response of the C392X TRα1 mutant receptor to acetylation. We hypothesized that an aberrant response to acetylation is a characteristic of this particular RTH-α mutation. To test these hypotheses, HeLa cells were transfected with HA-tagged expression plasmids for TRα1 wild-type and C392X TRα1, and the transfected cells were used in cell fractionation, immunoprecipitation, and western blot procedures, as appropriate. HA-tagged expression plasmids for acetylation and nonacetylation mimics of TRα1 wild-type and C392X TRα1 were also used to compare and validate the findings. The results showed that there is a higher population of acetylated TRα1 wild-type in the nucleus than in the cytoplasm, however, acetylated TRα1 in the nucleus is very susceptible to protease activity. Additionally, the TRα1 acetylation mimics showed increased cytosolic localization compared to the TRα1 nonacetylation mimics, which supports our hypothesis. Finally, in terms of C392X TRα1, our hypothesis was not supported as the mutant receptors did not show any apparent subcellular mislocalization or altered response to acetylation. This study further supports a role of acetylation in TRα1 nucleocytoplasmic shuttling, and provides new data for continued research to fully comprehend the role of acetylation in the proper functioning of the thyroid hormone receptor.