Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptides

Abstract

The delivery of entire functional proteins into living cells is a long-sought goal in science. Cyclic cell-penetrating peptides (cCPPs) have proven themselves to be potent delivery vehicles to carry proteins upon conjugation into the cytosol of living cells with immediate bioavailability via a non-endosomal uptake pathway. With this strategy, we pursue the cytosolic delivery of mCherry, a medium-sized fluorescent protein. Afterward, we achieve subcellular delivery of mCherry to different intracellular loci by genetic fusion of targeting peptides to the protein sequence. We show efficient transport into a membrane-bound compartment, the nucleus, as well as targeting of the actin cytoskeleton, marking one of the first ways to label actin fluorescently in genetically unmodified living cells. Furthermore, we demonstrate that only by conjugation of cCPPs via a disulfide bond, is flawless localization to the target area achieved. This finding underlines the importance of using a cCPP-based delivery vehicle that is cleaved inside cells, for the precise intracellular localization of a protein of interest.

Head to tail cyclisation of cell-penetrating peptides: impact on GAG-dependent internalisation and direct translocation

“…9 Among backbone modifications, cyclisation has been found to give remarkable results and is increasingly applied to CPPs. [10][11][12][13][14][15] In particular, cyclisation of Tat was found to improve cellular uptake by inducing maximal spacing between the CPP guanidinium groups, 11 in agreement with earlier observations made with peptoid analogues of CPPs. 16 Lipidation is another interesting modification, which can improve CPP interaction with the cell membrane leading to increased translocation efficiency.…”

Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization

“…Therefore, we aimed for the cyclization of an R 10 peptide, which is, like other arginine-rich peptides, able to enhance the transduction of functional proteins into living cells when covalently attached to the protein cargo 48. We and others observed that the cellular transduction behavior is significantly improved for the cyclic version of cR 10 when compared to its linear counterpart 22,49,50. As an alternative to the previously applied cyclized R 10 -CPP 14 , we synthesized peptide 15 containing a cysteine and an azidohomoalanine residue to replace the former lactam bridge after our novel cyclization technique.…”

Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

“…We have recently demonstrated the cleavage of peptides,bearing backbone Thz, using [PdCl(allyl)] 2 , [25,28] which is known to be compatible with reactions in live cells because of its reported low toxicity. [36] In addition, having one of the quenching pair on the CPP should allow loss of quenching upon Thzc leavage,r esulting in increased donor fluorescence. [33][34][35] Incorporation of backbone Thz, however,remains out of scope for genetic manipulation, making chemical protein synthesis essential for this task.…”