Chemoproteomic Profiling Reveals that Triiodothyronine Covalently Labels Cellular Proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Thyroid hormone triiodothyronine (T3) is essential for mammalian development and metabolism.
• T3's actions are traditionally understood through non-covalent interactions.
• A novel T3-based photoaffinity probe was developed to identify T3-interacting proteins.

Purpose of the Study:

• To investigate T3's interaction with cellular proteins.
• To identify novel T3-binding proteins and characterize the mechanism of interaction.
• To challenge the classical understanding of thyroid hormone signaling.

Main Methods:

• Synthesis of novel T3-based photoaffinity probes, including fluorescein-modified FIT3.
• Chemical proteomics using CO/IP combined SILAC to profile covalently labeled proteins.
• Activity-based protein profiling with alkyne-functionalized T3 probes and click chemistry for target identification.

Main Results:

• T3 was found to covalently bind to cellular proteins independently of photoirradiation.
• Site-mapping identified cysteine residues as modification sites via nucleophilic attack with iodine displacement from T3.
• Over 1000 candidate proteins, including ATP1A1, HSP90AB1, and PRDX1, were identified as covalent T3 targets, with validation by Western blotting.

Conclusions:

• Thyroid hormone action involves a previously unrecognized mode of covalent protein modification.
• This covalent interaction challenges the classical paradigm of thyroid hormone signaling.
• The findings offer new insights into hormone biology and identify potential therapeutic targets.