Delivery of antibodies to the cytosol: Debunking the myths

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG₁ Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells.     

Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects

We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 23-50 (mPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced ∼ 60% haemoglobin leakage after 30 min, whereas mPrPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be ∼ 12% for 50 μM mPrPp(1-28), and ∼ 1% for 50 μM mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.     

Novel Rath peptide for intracellular delivery of protein and nucleic acids

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30 min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant β structure with a small α helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.     

A cell-penetrating helical peptide as a potential HIV-1 inhibitor

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer α-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 Å. Using this structural information, we have utilized a structure-based rational design approach to stabilize the α-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced α-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.     

Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 °C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.     

Structure-activity relationship study of Aib-containing amphipathic helical peptide-cyclic RGD conjugates as carriers for siRNA delivery

The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp-d-Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide (PI) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure–activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus (PII) and both the N- and C-termini (PIII) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 (PIV), 12 (PV), and 8 (PVI) amino acid residues at the N-terminus of the helical chain were synthesized. PII and PIII, as well as PI, could deliver anti-luciferase siRNA into cells to induce the knockdown of luciferase stably expressed in cells. In contrast, all of the truncated peptides were unlikely to transport siRNA into cells.     

Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells

Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes.Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanismleading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases(also termed caspases) have been discovered and implicated as the common effectors of apoptosis. Inthis report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectruminhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events werefollowed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetictetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, suchas morphological characteristics and DNA fragmentation. These results suggest that staurosporine-inducedapoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.     

Aib-containing peptide analogs: Cellular uptake and utilization in oligonucleotide delivery

α-Aminoisobutyric acid (Aib)-containing peptide analogs derived from TV-XIIa, a cell-penetrating peptide (CPP), were synthesized to explore structure-activity relationships. The replacement of Aib at position 1, 5, or 9 in the TV-XIIa amino acid sequence with alanine (Ala) suppressed the cellular uptake, whereas the simultaneous substitution of the two proline (Pro) residues at positions 6 and 10 with Aib (P-IV) considerably increased the cellular uptake. In order to explore the potential use of the Aib-containing peptide analogs for the cellular delivery of oligonucleotides (ODNs), we synthesized a covalent conjugate (P-IV-AON) of a 15-mer antisense ODN, which is complementary to luciferase gene, with P-IV, and the antisense effect of the P-IV-AON conjugate on luciferase expression in A549 cells was examined. Luciferase expression was decreased in the presence of the conjugate upon treatment with the reaction buffer at the concentrations of 5 and 10 μM.     

Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.     

Fluorescent sterols monitor cell penetrating peptide Pep-1 mediated uptake and intracellular targeting of cargo protein in living cells

Although cell-penetrating peptides (CPP) facilitate endocytic uptake of proteins, little is known regarding the extent to which CPPs facilitate protein cargo exit from endocytic vesicles for targeting to other intracellular sites. Since the plasma membrane and less so intracellular membranes contain cholesterol, the fluorescent sterol analogues dansyl-cholestanol (DChol) and dehydroergosterol (DHE) were used to monitor the uptake and intracellular distribution of fluorescent-tagged acyl coenzyme A binding protein (ACBP) into COS-7 cells and rat hepatoma cells. Confocal microscopy colocalized DChol and Texas Red-ACBP (TR-ACBP) with markers for the major endocytosis pathways, especially fluorescent-labeled cholera toxin (marker of ganglioside GM1 in plasma membrane lipid rafts) and dextran (macropinocytosis marker), but less so with transferrin (clathrin-mediated endocytosis marker). These findings were confirmed by multiphoton laser scanning microscopy colocalization of TR-ACBP with DHE (naturally-fluorescent sterol) and by double immunofluorescence labeling of native endogenous ACBP. Serum greatly and Pep-1 further 2.4-fold facilitated uptake of TR-ACBP, but neither altered the relative proportion of TR-ACBP colocalized with membranes/organelles (nearly 80%) vs cytoplasm and/or nucleoplasm (20%). Interestingly, Pep-1 selectively increased TR-ACBP associated with mitochondria while concomitantly decreasing that in endoplasmic reticulum. In summary, fluorescent sterols (DChol, DHE) were useful markers for comparing the distributions of both transported and endogenous proteins. Pep-1 modestly enhanced the translocation and altered the intracellular targeting of exogenous-delivered (TR-ACBP) in living cells.