Transduction of peptides and proteins into live cells by cell penetrating peptides

Abstract

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.

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

“…Most of these applications use CPPs (Tat, penetratin, polyarginine, VP22) covalently linked to peptides or as fusion proteins (Mussbach et al, 2011;Shin et al, 2014).…”

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

“…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]. The studies on the intracellular distribution of CPPs in vivo are relatively rare [17,19,37]. Recently, Tsien et al [38,39] have reported the intracellular distribution of D-Arg oligomers in tissue cells, and they claimed that D-Arg oligomers were trapped within endocytic vesicles rather than localized in the cytosol in the cells of organs after injecting mice intravenously (IV) with the maximum tolerated dosage, regardless of whether the CPP was injected alone or attached to a polyanionic domain.…”

A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape

“…Although several researchers reported the intracellular delivery of proteins by CPPs [33][34][35], the biological activities of the proteins in the cytosol were often indistinct, possibly due to the low efficiency of endosomal escape. Even when intracellular delivery was improved by FPs, previous studies generally revealed the cytosolic localization of cargo proteins [17] but could not reveal their activities, except for some enzymes such as Cre recombinase [16], for which only a small amount is needed to analyze the activity.…”