Identification and Characterization of a New Family of Cell-penetrating Peptides

Abstract

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine

“…On the contrary, kalata B1, by targeting phosphatidylehanolaminephospholipids, interacts directly with the cell membrane. SFTI-1 does not interact with any of the phospholipids, and its mechanism of penetration appears to be distinct from McoTI-II and kalata B1 (Cascales et al, 2011;Nawae et al, 2014).…”

Degradation of an Old Human Protein

“…These include ␤A3-crystallin 188 -215 and ␣B-crystallin 1-18 and 2-18. All of these peptides also contain an RR sequence that is known to bind tightly to the phosphate headgroup of various membrane phospholipids (56,57). The binding of these peptides to the membrane may act as a "seeding point" and facilitate crystallin association with the peptides on the membrane surface.…”

Identification of Adenovirus-Derived Cell-Penetrating Peptide

“…24) Furthermore, CPPs composed of hydrophobic amino acids have also been reported, and various peptides are used as CPPs. 25) Peptide C has no bias in certain positively charged amino acid residues and interacts with trypsin-sensitive molecules on the surface of K562 cells. In previous studies, various CPPs had different capacities for internalizing through the plasma membrane, with some capable of penetrating bio-membranes, such as the Blood-Brain Barrier, the gastral membrane and the skin.…”