Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics

Mitochondria are attractive therapeutic targets for develop- ing agents to delay age-related frailty and diseases. However, few promising leads have been identified from natural products. Previously, we identified roseltide rT1, a hyperstable 27-residue cysteine-rich peptide from Hibiscus sabdariffa, as a knottin- type neutrophil elastase inhibitor. Here, we show that roseltide rT1 is also a cell-penetrating, mitochondria-targeting peptide that increases ATP production. Results from flow cytometry, live-cell imaging, pulldown assays, and genetically-modified cell lines supported that roseltide rT1 enters cells via glycosamino- glycan-dependent endocytosis, and enters the mitochondria through TOM20, a mitochondrial protein import receptor. We further showed that roseltide rT1 increases cellular ATP pro- duction via mitochondrial membrane hyperpolarization. Using biotinylated roseltide rT1 for target identification and pro- teomic analysis, we showed that human mitochondrial mem- brane ATP synthase subunit O is an intramitochondrial target. Collectively, these data support our discovery that roseltide rT1 is a first-in-class mitochondria-targeting, cysteine-rich peptide with potentials to be developed into tools to further our under- standing of mitochrondria-related diseases.