Crosslinking-Assisted Substrate Identification for Lysine Demethylases Abstract. Reversible lysine methylation on histone proteins constitutes a primary mechanism for gene regulation. Of particular importance is the oxidative removal of the methyl groups by a conserved family of Fe2+- and 2-ketoglutarate-dependent lysine demethylases (KDMs) that significantly affect transcriptional potential of a gene. However, gene regulatory activity of KDMs is inherently complex due to their ability to demethylate a wide range of non-histone proteins. This raises an important question: Are the biological functions of a given KDM manifested through its histone or non-histone substrates or both? Currently, no method exists to characterize KDM substrates in proteome-wide manner. To circumvent the challenge, we propose to develop a novel chemoproteomic approach termed ?crosslinking-assisted substrate identification (CASI)?. The CASI platform involves engineering of a KDM active site with a photosensitive amino acid for light-mediated crosslinking with the bound substrates followed by identification of the crosslinked species using quantitative mass spectrometry. Using development- and cancer-relevant lysine demethylase 4A (KDM4A) as paradigm, we show that such engineering approach is feasible. We plan to extend this approach to all the members of the KDM4 family and to identify their distinct substrates from human cells. Subsequent biochemical studies of the newly identified non- histone substrates would lead to improved understanding of how KDM4-mediated demethylation of critical cellular proteins controls protein-protein interactions, reprograms gene expression, repairs DNA damage and promotes tumor metastasis. We anticipate the CASI approach to be highly general and applicable to any chromatin-modifying enzyme to elucidate their functions in a manner not attainable by existing methods.
Relevancy Statement. Overexpression, point mutation and translocation in histone demethylases are common to multiple pathological conditions such as autoimmune diseases and cancer. The current proposal outlines a chemoproteomic platform for understanding how biochemical deregulation of these enzymes leads to human diseases. The successful implication of the proposed work is expected to advance our understanding of such aberrant processes and identify novel protein targets for drug development. !