Intrinsically Cell-Permeable Miniature Proteins Based on a Minimal Cationic PPII Motif

Abstract

Cell-penetrating peptides (CPPs) provide promising tools for the cellular delivery of molecular cargos ranging in size from small molecules and peptides to proteins and quantum dots. CPPs are typically cationic and/or amphipathic sequences that are unstructured or alpha-helical. We expand the repertoire of cell-penetrating motifs by designing encodable CPPs possessing type-II polyproline (PPII) helical structure. These motifs surpass the uptake efficiency of existing CPPs and are not cytotoxic at concentrations 100 times greater than that necessary for delivery. By replacing the PPII helix of a miniature protein, the motif can endow intrinsic cell permeability without increasing molecular size.

Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues

“…Several CPPs, such as D-Arg oligomers, (PRR) 5 (P: proline, R: L-Arg ), oligomers containing α-aminoxy acids, β-arginine oligomers, MPG, Pep, and CADY [29][30][31][32][33][34][35], have been observed to be localized in the cytosol in cultured cells at low micromolar concentrations and some of these CPPs have been successfully applied for in vivo delivary. However, most of the in vivo studies of these and other CPPs were focused on the tissue distributions or bioactivity of CPPs and their cargoes [35,36].…”

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

“…When the replacement residues are all positively or all negatively charged, the resulting ''supercharged'' proteins can retain their activity while gaining unusual properties such as robust resistance to aggregation and the ability to bind oppositely charged macromolecules. For example, we reported that a green fluorescent protein with a ϩ36 net theoretical charge (ϩ36 GFP) was highly aggregation-resistant, could retain fluorescence even after being boiled and cooled, and reversibly complexed DNA and RNA through electrostatic interactions.A variety of cationic peptides and proteins have been observed to penetrate mammalian cells (18)(19)(20)(21)(22)(23)(24). We hypothesized that superpositively charged proteins such as ϩ36 GFP might also associate with negatively charged components of the cell membrane in a manner that results in cell penetration.…”

“…A variety of cationic peptides and proteins have been observed to penetrate mammalian cells (18)(19)(20)(21)(22)(23)(24). We hypothesized that superpositively charged proteins such as ϩ36 GFP might also associate with negatively charged components of the cell membrane in a manner that results in cell penetration.…”

Comparative analysis of the methods of drug and protein delivery for the treatment of cancer, genetic diseases and diagnostics

“…Non-toxic secretory RNases are converted to highly cytotoxic ones by cationization with ethylenediamine (Futami, 2005). A variety of cationic peptides and proteins have been observed to penetrate mammalian cells (Lawrence, 2007;McNaughton, 2009;Frankel, 1988;Green, 1988;Thoren, 2000;Daniels, 2007;Smith, 2008;. Peptoids are designed of high stability and resistant to proteolysis, transporting macromolecules into cells by offering a highly efficient, non-toxic and non-immunogenic route for cell entry.…”