Discovery of a novel player in ER-Mitochondria interactions by DARTS-LC-MS/MS proteomics analysis

Mitochondria-associated ER membranes (MAMs) have attracted attention for their important roles in physiological processes such as calcium transport, lipid metabolism, and organelle dynamics. In this study, we observed increased ER-mitochondria contacts and impaired autophagy in vascular endothelial cells stimulated by oxidized low-density lipoprotein (oxLDL) and in the apoE-/- mouse model of atherosclerosis. We found that CTS, a natural active compound , exhibited anti-atherosclerotic effects both in vitro and in vivo. In addition, CTS induced autophagy and reduced ER-mitochondria association. To elucidate the molecular mechanism of CTS underlying this effect, we performed DARTS-LC-MS/MS analysis combined with quantitative SWATH analysis to identify the target protein of CTS. A list of candidate targets showing more than 10% resistance to proteolysis upon CTS treatment was generated, revealing that most were localized to the ER or mitochondria. Based on high sequence coverage, we identified CTS-BP, an ER-resident chaperone protein, as the binding partner of CTS. Knockdown of the CTS-BP gene confirmed its role in mediating CTS effects, leading to MAM disruption and increased autophagy. Our findings provide novel insights into how an anti-atherosclerotic natural product modulates ER-mitochondria interactions to promote autophagy for its pharmacological activity.