The Effect Of Acetylation On The Stability And Ubiquitin-Mediated Degradation Of Trɑ1

Thyroid hormone receptor (TRɑ1) is a nuclear receptor involved in the transcriptional regulation of thyroid hormone-responsive genes. Its mechanism of action is characterized by post-translational modifications. Lysine acetylation of TRɑ1 plays a central role in activating target genes. It is hypothesized that acetylation also modulates the nucleocytoplasmic shuttling of TRɑ1, as key acetylation sites occur in its nuclear localization signal (Mavinakere et al., 2012; Sánchez-Pacheco et al., 2009). Additionally, localization studies demonstrated that acetylated TRɑ1 has a higher cytoplasmic population, while nonacetylated TRɑ1 has higher nuclear retention (Anyetei-Anum et al., 2019). As a transcription factor, the nuclear function of TRɑ1 is clear, however, its role in the cytoplasm is not well established (Anyetei-Anum et al., 2018). Considering that TRɑ1 is both acetylated and ubiquitinated (Bondzi et al., 2011; Dace et al., 2000; Kenessey & Ojamaa, 2005; Subramanian et al., 2015), it may be deacetylated in the cytoplasm or polyubiquitinated and targeted to the proteasome for degradation. To further characterize the role of acetylation and uncover the cytoplasmic role of TR, this thesis aimed to qualitatively assess the impact of acetylation on the stability and ubiquitin-mediated degradation of TRɑ1. The relative impact of acetylation on TRɑ1 stability was evaluated by performing a cycloheximide chase assay using HeLa cells transfected with wildtype TRɑ1, and its acetylation and nonacetylation mimics. After a 10-hour chase, we found no statistically significant difference in expression levels at select time points between all TR variants. Next, we investigated the effect of acetylation on the ubiquitination of TRɑ1 by utilizing immunoprecipitation and western blot analysis, in the absence of proteasome inhibitors. We showed that TRɑ1 and its acetylation mimic exhibit a polyubiquitin signal, while the nonacetylation mimic was monoubiquitinated in low amounts. Proteins targeted for proteasome-mediated degradation are tagged with a polyubiquitin chain due to their reduced stability, but polyubiquitination may also be attributed to proteasome-mediated transcriptional regulation. Since we found that acetylation did not reduce TRɑ1 stability, the polyubiquitin signal detected may have a non-proteolytic function. These results suggest that acetylation and ubiquitination may work in tandem to modulate proteasomal-mediated transcriptional regulation. However, further studies must be carried out to confidently attribute the polyubiquitination of acetylated TRɑ1 to transcriptional regulation or degradation. This research has implications for understanding how post-translational modifications contribute to the functionality of TRɑ1 and mutant variants that cause Resistance to Thyroid Hormone Syndrome.