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 IκB𝛼 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.