Hyperstable Cell‐Penetrating Peptides from Medicinal Plants

A longstanding interest of our laboratory is to study cysteine‐rich peptides (CRPs) from medicinal plants as an inspiration to design orally‐active compounds. Plants produce CRPs as part of their host‐defense mechanism against microbes and insects. Most CRPs contain 15 to 25% of cysteine per molecule and are characterized by their cystine scaffold [1– 4]. CRPs can display both hydrophilic and hydrophobic surface patches due to the cystine dense core which causes hydrophobic side chains to flip outwards, resulting in a unique “inside‐out” feature. As hydrophobicity is a major driving force for cell penetration [5], we hypothesized that CRPs penetrate cells and bind to intracellular targets to exert their biological effects. Here, we report the discovery of hyperstable and cell‐penetrating CRPs, roseltide rT1 and rT7, from Hibiscus sabdariffa of the Malvaceae family. We show that both roseltide rT1 and rT7 are highly resistant against proteolytic degradation. Using live‐cell confocal microscopy, we show that cationic roseltide rT1 and anionic roseltide rT7 are cell‐penetrating. Functionally, we demonstrate that roseltide rT1 targets the mitochondria via TOM20 and increases cellular ATP production [4]. Roseltide rT7, on the other hand, inhibits the 20S proteasome, which in turn, reduces tumor necrosis factor‐induced IκB□ degradation, and decreases intercellular adhesion molecule‐1 expressions. Taken together, our results demonstrate that hyperstable CRPs are cell‐penetrating and targets intracellularly to elucidate their biological effects. Support or Funding Information This research was supported in part by Nanyang Technological University Internal Funding ‐ Synzymes and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).References [1] Tam , J. P. , Nguyen , G. K. , Loo , S. , Wang , S. , Yang , D. , & Kam , A. ( 2018 ). Ginsentides: Cysteine and Glycine-rich Peptides from the Ginseng Family with Unusual Disulfide Connectivity . Scientific reports , 8 ( 1 ), 16201 . [2] Loo , S. , Kam , A. , Xiao , T. , & Tam , J. P. ( 2017 ). Bleogens: cactus-derived anti-candida cysteine-rich peptides with three different precursor arrangements . Frontiers in plant science , 8 , 2162 . [3] Loo , S. , Kam , A. , Xiao , T. , Nguyen , G. K. , Liu , C. F. , & Tam , J. P. ( 2016 ). Identification and characterization of roseltide, a knottin-type neutrophil elastase inhibitor derived from Hibiscus sabdariffa . Scientific reports , 6 , 39401 . [4] Kam , A. , Loo , S. , Dutta , B. , Sze , S. K. , & Tam , J. P. ( 2019 ). Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics . Journal of Biological Chemistry , 294 ( 11 ), 4000 – 4011 . [5] Peraro , L. , & Kritzer , J. A. ( 2018 ). Emerging Methods and Design Principles for Cell-Penetrant Peptides . Angewandte Chemie International Edition , 57 ( 37 ), 11868 – 11881 .