Chemoproteomic Profiling Reveals that Triiodothyronine Covalently Labels Cellular Proteins

Thyroid hormone triiodothyronine (T3) is a critical regulator of mammalian development and metabolism, traditionally recognized for its actions. In this study, we initially designed and synthesized a novel T3-based photoaffinity probe in order to identify T3-interacting proteins in live cells. Remarkably, our results demonstrate that T3 can covalently bind to cellular proteins independently of photoirradiation. To validate this covalent labeling, a fluorescein-modified T3 probe (FIT3) 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 T3. Further, two activity-based probes bearing alkyne click handles at distinct positions on the T3 scaffold were further used to expand the profiling of covalent T3 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.