Page 7 - I. Chemical biology and drug discovery

Role of mitochondrial TUFM in alleviating metabolic dysregulation through enhancing autophagy
Dasol Kim , Yunyeong Jang , Hui-Yun Hwang , Eun Sun JI , Jin Young Kim , Jong Shin Yoo , and Ho Jeong Kwon *
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1 Chemical Genomics Global Research Laboratory, Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul 03722, Korea
2 Biomedical Omics Group, Korea Basic Science Institute, Ochang, Chungbuk 28119, Korea
*Corresponding author: kwonhj@yonsei.ac.kr
BACKGROUND AIM
Autophagy is a key regulatory mechanism for systemic homeostasis, To identify protein targets of K relevant with autophagy and
thus disorder of which causes a number of human metabolic diseases. metabolic improvement, we leveraged label-free drug affinity
Accordingly, discovery of new autophagy regulators and elucidation of responsive target stability (DARTS) approach with a liquid
underlying mechanism have been highly emphasized recently. We chromatography/tandem mass spectrometry (LC–MS/MS) readout.
identified a natural small molecule K as a autophagy inducer, which To validate the biological mechanism of K, we investigated
improves metabolic dysregulation. functional relevancy of target protein through genetic modulation.

METHODS

Combinational method of drug affinity responsive target stability (DARTS) with
LC−MS/MS identified a new protein target of compound K. (1) Exposure of the
small molecule to cell lysate proteome pool allowing formation of small molecule
– protein target interactions, (2) pronase digestion of proteins, where proteins
that underwent conformational change due to small molecule binding resist
against protease chopping while proteins with less or no binding to the small
molecule are degraded, (3) further proteolysis with trypsin and LC–MS/MS
(SWATH) analysis to determine differential peptide sequence, (4) identification
of proteins protected by the small molecule based on its enhanced peptide
amount more than 20% compared to controls, and (5) selection of protein target
candidates based on phenotypic relevancy to the small molecule.
RESULTS
Figure 1. Compound K induces autophagy and Figure 1 Figure 2
promotes degradation of lipid droplets (LDs) in
autophagy dependent manner. Autophagy dependent LDs clearance Metabolic improvement in obese mice
Figure 2. Compound K improves metabolic
dysregulation in obese mice.
Figure 3. Characterization of TUFM as a
potential protein target of Compound K.
Figure 4. Compound K requires TUFM to
promote LDs degradation, and over-expression
of TUFM alone enhances LDs clearance.
★ CQ : lysosomotrophic reagent
Figure 3 Figure 4
Peptide analysis detected in DARTS LC-MS DARTS biophysical validation Genetic modulation study

In silico docking validation

*PA/OA: fatty acid, lipid droplets loading reagents
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

Supported by BK21 PLUS, NRF (MSIP;
Collectively, our study revealed that Compound K [1] Chang, J., Kim, Y., and Kwon, H.J. Advances in 2015K1A1A2028365, 2018M3A9C4076477)
identification and validation of protein targets of natural
induces autophagy, and improves metabolic products without chemical modification. Nat. Prod. Rep. 2016,
regulations through directly binding to its target 33(5): 719-730.
protein TUFM. In addition to the role of Compound [2] Kim D., Hwang H.Y., Kwon H.J. Targeting Autophagy In
K as a modulator of TUFM, we also suggested a Disease: Recent Advances In Drug Discovery. Expert opin. Contact information
drug discov. 2020, 15(9):1045-1064.
new functional role of TUFM in metabolic
improvement. This novel role of TUFM likely [3] Hwang H.Y., Cho Y.S., Kim J.Y., et al. Autophagic Inhibition • Presenter: Dasol Kim
via Lysosomal Integrity Dysfunction Leads to Antitumor Activity
involves autophagy activation since it promotes in Glioma Treatment. Cancers 2020, 12(3): 543
autophahosome formation through mediating [4] Nunez, C.E., Rodrigues, V.S., Gomes, F.S., et al., Defective ekthf1728@yonsei.ac.kr
protein-protein interaction, and suggests a regulation of adipose tissue autophagy in obesity. Int. J. Obes. • Correspondence: Ho Jeong Kwon
2013, 37(11): 1473-1480.
mechanism of its biological action.
kwonhj@yonsei.ac.kr Presenter : Minjeong