Bioorthogonal chemical reporters for profiling retinoic acid-modified and retinoic acid-interacting proteins☆

Vitamin A and its primary active derivative, all-trans retinoic acid (RA), are endogenous signaling molecules essential for numerous biological processes, including cell proliferation, differentiation, and immune modulation. Owing to its differentiation-inducing effect, RA was the first differentiating agent approved for the clinical treatment of acute myeloid leukemia. While the classical mechanisms of RA signaling involve nuclear receptors, such as retinoic acid receptors (RARs), emerging evidence suggests that RA also engages in non-covalent and covalent interactions with a broader range of proteins. However, tools for thoroughly characterizing these interactions have been lacking, and a comprehensive understanding of the landscape of RA-modified and RAinteracting proteins remains limited. Here, we report the development of two RA-based chemical reporters, RA-yne and RA-diazyne, to profile RA-modified and RA-interacting proteins, respectively, in live cells. RA-yne features a clickable alkyne group for metabolic labeling of RA-modified proteins, while RA-diazyne incorporates a photoactivatable diazirine and an alkyne handle for crosslinking and capturing RA-interacting proteins. Using quantitative proteomics, we demonstrate the high-throughput identification of these proteins, revealing that non-covalent interactions are more prevalent than covalent modifications. Our global profiling also uncovers a large number of RA-interacting proteins mainly enriched in pathways related to mitochondrial processes, ER homeostasis, and lipid metabolism. Overall, this work introduces new RA-derived chemical reporters, expands the resource for studying RA biology, and enhances our understanding of RA-associated pathways in health and disease.