Co-operative membrane disruption between cell-penetrating peptide and cargo: implications for the therapeutic use of the Bcl-2 converter peptide D-NuBCP-9-r8.

Delivering apoptosis inducing peptides to cells is an emerging area in cancer and molecular therapeutics. Here, we have identified an alternative mechanism of action for the proapoptotic chimeric peptide D-NuBCP-9-r8. Integral to D-NuBCP-9-r8 is the Nur-77-derived D-isoform sequence fsrslhsll that targets Bcl-2, and the cell-penetrating peptide (CPP) octaarginine (r8) that is required for intracellular delivery. We find that the N-terminal phenylalanine of fsrslhsll acts in synergy with the cell-penetrating moiety to enhance peptide uptake at low nontoxic levels and cause rapid membrane blebbing and cell necrosis at higher (IC(50)) concentrations. These effects were not observed when a single phenylalanine-alanine mutation was introduced at the N-terminus of D-NuBCP-9-r8. Using primary samples from chronic lymphocytic leukemia (CLL) patients and cancer cell lines, we show that NuBCP-9-r8 induced toxicity, via membrane disruption, is independent of Bcl-2 expression. Overall, this study demonstrates a new mechanism of action for this peptide and cautions its use as a highly specific entity for targeting Bcl-2. For delivery of therapeutic peptides the work emphasizes that key amino acids in cargo, located several residues away from the cell-penetrating sequence, can significantly influence their cellular uptake and mode of action.

Chemical synthesis of cell-permeable apoptotic peptides from in vivo produced proteins.

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.

Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide.

The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 microM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the suspension cells studied, but a 10-fold higher concentration was required to observe similar results in adherent cells. At 37 degrees C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of a requirement for nucleation zones to promote this effect. Cholesterol depletion with methyl-beta-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 degrees C, but decreased overall peptide accumulation. Under the same conditions in adherent cells this agent had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide at 4 degrees C in KG1a cells, but this effect could be reversed by re-addition of cholesterol as methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially at low temperatures, suggesting that this feature could be exploited for delivering bioactive entities.

BH3 helix-derived biophotonic nanoswitches regulate cytochrome c release in permeabilised cells.

Dynamic physical interactions between proteins underpin all key cellular processes and are a highly attractive area for the development of research tools and medicines. Protein-protein interactions frequently involve α-helical structures, but peptides matching the sequences of these structures usually do not fold correctly in isolation. Therefore, much research has focused on the creation of small peptides that adopt stable α-helical structures even in the absence of their intended protein targets. We show that short peptides alkylated with azobenzene crosslinkers can be used to photo-stimulate mitochondrial membrane depolarization and cytochrome c release in permeabilised cells, the initial events of the intrinsic apoptosis pathway.

Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence PFVYLI linked to the proapoptotic domain peptide PAD.

The capacity of cell penetrating peptides (CPPs) to breach biological membranes offers hope for their utilisation as vectors for the delivery of small molecule drugs and macromolecular therapeutics. Using three different cell systems, including primary human cells, we have studied the uptake, subcellular localisation and effect on cell viability of the well characterised octaarginine and the more recently discovered hydrophobic PFVYLI peptide, either alone, or conjugated to the proapoptotic domain peptide PAD (klaklak)(2). Octaarginine and PFVYLI were efficiently endocytosed into cells at 37 degrees C but an ability to translocate directly across the plasma membrane at higher peptide concentrations or when uptake experiments were performed on ice was confined to the cationic variant. Octaarginine- and PFVYLI-PAD conjugates were cytotoxic, with KG1a leukaemia cells being more sensitive than HeLa cells and octaarginine-PAD being the most potent conjugate in both cell lines. The effects of the CPP-PAD conjugates on cell morphology and permeability was rapid suggesting that cytotoxicity is partially mediated at the plasma membrane rather than exclusively through induction of apoptosis at the mitochondria. Primary human leukaemia cells were more similar to KG1a cells than HeLa cells, suggesting the relative sensitivity of leukaemia cells to these peptides could be exploited in vivo.