Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

The structure of the cell-permeable alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 microM. These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non-amphipathic 18-mer peptides with different primary structure, net charge and helix parameters from I. The amphipathic counterparts were internalized into the cells to a comparable extent as I, whereas no cellular uptake could be detected for the non-amphipathic analogues. The mode of uptake remains unclear and involves both temperature-sensitive and -insensitive processes, indicating non-endocytic contributions.

The cellular uptake of a peptide set derived from membrane-permeable a-helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity-dependent differences became apparent only after washing the peptide-loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 8C led to similar results, indicating the nonendocytic character of the uptake.With a view to practical applications, the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell-type specific vector peptides.Keywords: amphipathic peptides; cell-penetrating peptides; cellular uptake; confocal laser scanning microscopy.Polar biopolymers, such as oligonucleotides or peptides, are becoming increasingly important as highly specific, intracellularly active biochemical agents or potential therapeutics. The value of such compounds, however, is compromised by their limited ability to cross the plasma membrane. Most nondestructive strategies for translocating these biopolymers into the cell interior rely on endocytic mechanisms, and therefore suffer from poor and unreliable delivery into the cytosol or nucleus, the main target compartments. As promising alternatives, nonendocytic translocation approaches, based on the use of natural leader peptide derivatives, have been presented recently [1±8]. These peptides proved to be suitable for the translocation of covalently tagged oligopeptides or oligonucleotides across biological membranes, thereby circumventing the problems of an endocytic mode of uptake. The nonendocytic nature of the membrane passage is indicated by the observation that efficient translocation occurs even at 0 8C; however, the actual mechanism remains unclear.In previous studies, we were able to mimic the permeation behavior of the aforementioned natural peptides with simple helical amphipathic model peptides [9±11]. Our results indicated that multiple, energy-dependent and -independent mechanisms are involved. After alteration of the peptide structure, we found cellular uptake only for am...

SummaryExtracellular administration of a membrane permeable model peptide containing the tripeptide sequence, SLV, at the C-terminus to human endothelial and kidney cells resulted in an induction of caspase-8 (FLICE), the apical enzyme of the apoptosis cascade. The unmodified or N-terminally SLV-tagged peptide had no effect, thereby eliminating an unspecific induction of apoptosis as the cause of the caspase activity observed. Drastic alterations of primary structure and structure forming properties of the carrier peptide did not significantly influence the caspase-8 inducing activity of the C-terminal SLV-tag, supporting previous findings that translocation into the cell interior is a more general ability of peptides.

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