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CPTH2-Driven Acetyltransferase Inhibition Impacts Thyroid Hormone Receptor Localization and Acetylation Levels
Rosenthal, Matthew I
Rosenthal, Matthew I
Abstract
Thyroid hormone plays a critical role in regulating metabolism, growth, and development by binding to the thyroid hormone receptor (TR) and modulating gene expression. Evidence indicates an elaborate system of TR nuclear import and export that extends beyond its role as a transcription factor. TR contains nuclear localization signals (NLSs) and nuclear export signals (NESs) that promote interactions with nuclear transport proteins and subsequent import or export through nuclear pore complexes. The precise mechanisms and reasonings underlying TR nucleocytoplasmic shuttling remain elusive. A working model suggests the involvement of a series of acetylation events catalyzed by lysine acetyltransferases (KATs). However, it remains unclear which particular KATs are involved. Our previous work with C646, a specific inhibitor of the CBP/p300 KAT, points to this enzyme’s involvement in TR nucleocytoplasmic shuttling. To investigate the involvement of other KATs, I utilized CPTH2, a broad-spectrum inhibitor of KATs GCN5, CBP/p300 and, to a lesser degree, PCAF. After transfection with GFP-tagged TRα1, TRβ1, or TRβ2 expression plasmids, CPTH2-inhibited HeLa (human) cells were tested against dimethyl sulfoxide (DMSO) (vehicle)-control cells. Cells were visualized using fluorescence microscopy, and nuclear-to-cytoplasmic fluorescence ratios were calculated and averaged for each treatment group. Since TRα1 is already predominantly localized to the nucleus, the addition of CPTH2 did not detectably affect localization. However, the CPTH2-inhibited TRβ1 and TRβ2 groups showed significantly reduced cytoplasmic localization compared to the DMSO-controls, with p-values of 1.86 × 10-95 and 5.20 × 10-95, respectively. Phalloidin-TRITC staining of actin also revealed improper morphology and decreased cell-cell communication at higher concentrations of CPTH2. This change was not observed in cells treated with C646, implying GCN5 is involved in the maintenance of actin stress fibers. Changes in TR acetylation levels between CPTH2 and DMSO groups were determined by immunoprecipitation. Lysates from HeLa cells transfected with HA-tagged TRα1 were processed with acetyl-lysine affinity beads and analyzed by immunoblotting with anti-HA antibodies. While CPTH2-inhibited cells did show less acetylated TR compared to the DMSO-control on average, results were variable, and the p-value for this experimentation was not significant (0.191). Further localization experimentation compared treatment with CPTH2 versus C646 to specifically investigate whether GCN5 interacts with TR in addition to CBP/p300. HeLa cells transfected with GFP-tagged TRβ1 were treated with CPTH2, C646, or DMSO (vehicle)-control. Statistical analysis between the CPTH2 and C646 treatments yielded a p-value of 0.144, indicating CPTH2 inhibits proper TR localization comparably to C646. TRα1 immunoprecipitation and subsequent detection by α-GCN5 antibody did not reveal any interaction between the two proteins. It remains unclear whether GCN5 is directly implicated in the acetylation of TR and its nucleocytoplasmic shuttling mechanism. While CPTH2 global inhibition superficially points to involvement, C646 comparative analysis and TRα1 co-immunoprecipitation contradict this claim. Regardless, the results of our experimentation further advance our understanding of the acetylation-dependent model of TR shuttling and may highlight potential targets for therapeutic intervention in diseases linked to improper TR localization.
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2025-05-01
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5/6/2027
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Biology