Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation.

Disulfide-rich plant peptides with molecular weights of 2 to 6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue-peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family), that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, 6C-hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is > 100-fold more stable against protease degradation than its S-alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBᵯC; degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies.