Chemoproteomic Profiling Reveals that Triiodothyronine Covalently Labels Cellular Proteins

Thyroid hormone triiodothyronine (<b>T3</b>) is a critical regulator of mammalian development and metabolism, traditionally recognized for its actions. In this study, we initially designed and synthesized a novel <b>T3</b>-based photoaffinity probe in order to identify <b>T3</b>-interacting proteins in live cells. Remarkably, our results demonstrate that <b>T3</b> can covalently bind to cellular proteins independently of photoirradiation. To validate this covalent labeling, a fluorescein-modified <b>T3</b> probe (<b>FIT3</b>) was utilized, and a CO/IP combined SILAC approach was applied to profile covalently labeled proteins. Focusing on one putative target, succinate dehydrogenase subunit A (SDHA), site-mapping analysis identified cysteine residues as likely covalent modification sites mediated by a nucleophilic reaction through iodine leaving from <b>T3</b>. Further, two activity-based probes bearing alkyne click handles at distinct positions on the <b>T3</b> scaffold were further used to expand the profiling of covalent <b>T3</b> targets. This approach uncovered over 1000 candidate proteins, including ATP1A1, HSP90AB1, and PRDX1, with selected targets validated by Western blotting. These findings reveal a previously unrecognized mode of thyroid hormone action involving covalent protein modification, challenging the classical paradigm of thyroid hormone signaling and offering new insights into hormone biology and potential therapeutic targets.