Abstract
Mitochondria are attractive therapeutic targets for developing 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 glycosaminoglycan-dependent endocytosis, and enters the mitochondria through TOM20, a mitochondrial protein import receptor. We further showed that roseltide rT1 increases cellular ATP production via mitochondrial membrane hyperpolarization. Using biotinylated roseltide rT1 for target identification and proteomic analysis, we showed that human mitochondrial membrane 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 understanding of mitochrondria-related diseases.
Highlights
Mitochondria are attractive therapeutic targets for developing agents to delay age-related frailty and diseases
Natural and synthetic roseltide rT1 were identical as determined by MALDI-TOF mass spectrometry (MS), co-elution by RP-high-performance liquid chromatography (LC) (HPLC), and overlay of their two-dimensional NOESY spectra
The results showed that cyanine 3 (Cy3)-rT1 colocalized with TOM20-green fluorescent protein (GFP) in vitro (Fig. 4C, Videos S1 and S2) and that mitochondria uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) did not inhibit the mitochondrial localization properties of Cy3-labeled roseltide rT1 (Cy3-rT1) (Fig. 4D)
Summary
Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics. Using biotinylated roseltide rT1 for target identification and proteomic analysis, we showed that human mitochondrial membrane ATP synthase subunit O is an intramitochondrial target 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 understanding of mitochrondria-related diseases. Roseltide rT1 shares certain biophysical features similar to mitochondria-targeting molecules and Leu/Ile-rich helical peptides. It is positively-charged, has a Leu/Ile-rich sequence with 85% hydrophobic amino acids residues (Fig. S1), and four inter-cysteine loops that form a structure that resembles a four-leaf clover. Roseltide rT1 could represent a novel class of naturally occurring mitochondria-targeting CRPs (mtCRP) with no sequence homology or structural similarity to known mitochondria-targeting helical peptides. Our findings highlight the characterization of a first-in-class, hyperstable, plant-derived mtCRP, which represents a promising lead to increase the health span of aging populations
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