Program

Poster Presentations

Day 2, May 16（Wed.）　　Poster

2P-14　　PDF

Identification of protein targets of a bioactive small molecule with DARTS combined with LC-MS/MS proteome analysis

(¹Yonsei University, ²Severance Biomedical Science, ³Korea Basic Science)
^oDongjin Lee¹, Hui-yun Hwang¹, Myung-shik Lee², Hyejin Lim², Jin Young Kim³, Jong Shin Yoo³, Ho Jeong Kwon¹

Identification of protein target of bioactive small molecules provides the basis for the understanding of its mode of actions. We have developed a new label-free target protein identification method by combination of DARTS (Drug Affinity Responsive Target Stability) with LC-MS/MS. Recently, we found out a new small molecule (MSL) enhancing autophagy activity through TFEB (transcription factor EB) translocation and identified Calcineurin A (CaN)as a protein target of MSL through DARTS assay. We further validated the specific interaction of MSL with CaN by comparing the binding activities of inactive derivatives of MSL to CaN. Through LC-MS/MS proteome analysis of the entire proteome treated with MSL with pronase, 20 proteins exhibited resistant to protease upon MSL treatment. Molecular and cellular investigations revealed that MSL could bind to other phosphatases like CaN for TFEB translocation into nucleus leading to activation of autophagy. These results provide the role of phosphatases in autophagy induction via TFEB regulation and combination of DARTS and LC-MS/MS method could be an efficient platform to identify protein targets of unmodified small molecules.