Targeting the 14-3-3θ protein with indole-3-propionic acid restores PINK1/Parkin mediated mitophagy and suppresses endothelial PANoptosis in atherosclerosis.

BACKGROUND: Indole-3-propionic acid (IPA) is a naturally occurring, microbiota-derived tryptophan metabolite; however, its vascular protective effects and the endothelial mechanisms involved in atherosclerosis remain unclear. PURPOSE: This study aimed to evaluate the anti-atherosclerotic efficacy of IPA and elucidate its endothelial targets and mechanisms underlying vascular protection. METHODS: In high-fat diet (HFD)-fed ApoE-/- mice, the anti-atherosclerotic effects of IPA were evaluated by en face aortic analysis, lipid deposition assessment, and collagen content quantification. In oxidized low-density lipoprotein (ox-LDL)-challenged human umbilical vein endothelial cells (HUVECs), PINK1/Parkin mediated mitophagy and PANoptosis-related signaling were examined by mitophagy flux assays and immunoblotting of pathway markers. Limited proteolysis-small molecule mapping/mass spectrometry (LiP-SMap/MS) and surface plasmon resonance (SPR) analysis were performed to profile IPA-interacting proteins, and adeno-associated virus (AAV)-mediated endothelial YWHAQ knockdown in ApoE-/- mice was used to validate target dependence. RESULTS: In HFD-fed ApoE-/- mice, oral IPA reduced en face aortic plaque burden, decreased lipid deposition and increased collagen content. In ox-LDL-challenged HUVECs, IPA enhanced PINK1/Parkin mediated mitophagy, improved mitochondrial function and mitigated PANoptosis. The 14-3-3θ protein (YWHAQ) was identified as a direct molecular target of IPA via LiP-SMap/MS and SPR analysis. Molecular docking predicted a Tyr48-centered binding pose on YWHAQ and substitution of Tyr48 with alanine markedly reduced IPA's cytoprotective effects. Mechanistically, ox-LDL weakened the YWHAQ-PINK1 association, which IPA restored in HUVECs. In vivo, AAV-mediated endothelial YWHAQ knockdown attenuated IPA's anti-atherosclerotic efficacy. CONCLUSION: These findings collectively indicate that IPA alleviates endothelial PANoptosis by engaging the 14-3-3θ protein to restore PINK1/Parkin mediated mitophagy and ultimately attenuates the progression of atherosclerosis.