Mitochondria‐targeting peptide from Hibiscus sabdariffa

Abstract

Cysteine‐rich peptides (CRPs) are hyperstable, multi‐disulfide‐constrained peptides. Structurally, CRPs contain evolutionary conserved disulfided scaffolds, often with functional intercysteine loops which contribute to their multiple biological activities. Previously, we have identified Hibiscus sabdariffa as a rich source of CRPs. Hibiscus sabdariffa, commonly known as roselle, is a South‐east Asian plant. It is used as a folk medicine for treating age‐related conditions and diseases. The prototypic CRP derived from roselle is roseltide rT1, a 27‐residue cysteine‐rich peptide with six cysteine forming four intercycteine loops. We showed that Loop 1 contributes to the inhibitory activities against human neutrophil elastase. However the functions of other loops, individually or in combination have not been determined. Here we report that roseltide rT1 is a mitochondria‐targeting peptide with bioenergetic properties. We showed that roseltide rT1 interacts with the mitochondrial import receptor TOM20 for its localization to the mitochondria. We prepared the linear precursor of roseltide rT1 using a stepwise solid‐phase method followed by oxidative folding to its native state. The conformation of synthetic rT1 was confirmed using reversed‐phase high‐pressure liquid chromatography and nuclear magnetic resonance spectroscopy. Using N‐hydroxysuccinimide ester, N‐terminal biotin‐ and Cy3‐labeled roseltide rT1 were generated for biological assays. Our results showed that mitochondrial depolarization induced by mitochondria uncoupler carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone does not abolish the mitochondrial localization of Cy3‐rT1. In silico docking suggested that the leucine‐rich loop 2 of roseltide rT1 interacts with TOM20. Using pull‐down assay, biotin‐rT1 was shown to interact with TOM20. Using live‐cell confocal microscopy, Cy3‐rT1 was shown to co‐localize with TOM20‐GFP in stably‐transfected HEK293 cells. Additionally, the localization of Cy3‐rT1 with MitoTracker Green was reduced in TOM20‐knocked‐down HepG2 cells compared to control. Taken together, this study shows that the mitochondrial import receptor TOM20 plays an important role for the localization of roseltide rT1 to the mitochondria.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.