Structural requirements for cellular uptake of α-helical amphipathic peptides

Abstract

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.

Pretreatment with PTD-Calbindin D 28k Alleviates Rat Brain Injury Induced by Ischemia and Reperfusion

“…The mechanism of PTD-mediated protein transduction, independent of energy and receptors binding, has not yet been fully elucidated. A nonspecific electrostatic interaction of basic amino acids PTD with cellular membrane might be the first crucial step (Bellet-Amalric et al, 2000); heparan sulfate in the cytoplasm (Rusnati et al, 1999;Ziegler and Seelig, 2004) and amphiphilic a-helices of PTD might be required during the transduction (Scheller et al, 1999). For the hippocampal slices, we obtained a similar result; after incubation with FITC-labeled PTD-CaBD, most of the cells showed FITC-positive staining.…”

Actin remodeling requires ERM function to facilitate AQP2 apical targeting

“…Nevertheless, the lack of six C-terminal amino acids completely prevents any actin binding (Turunen et al, 1994). To investigate the role of moesin in regulating the actin cytoskeleton in CD8 cells, a peptide corresponding to a highly conserved sequence in the ERM family at the C-terminal domain (ERM peptide) was synthesized and introduced into CD8 cells as described by Oehlke (Oehlke et al, 1998;Scheller et al, 1999) (Fig. 2A).…”

Peptide delivery to the brain via adsorptive-mediated endocytosis: Advances with SynB vectors

“…A number of other cell-penetrating peptides that derive not from natural proteins but from the engineering of various short peptides have been described, such as, for example, transportan, 10,20 the model amphipathic peptide (MAP), 16,17 and various signal sequence-based peptides, 11,12 and homoarginine vectors 34 (see Tables 1 and 2). Transportan is a 27-amino-acid chimeric peptide composed of the neuropeptide galanin and mastoparan-X linked by a central lysine.…”

“…10 Likewise, it has been reported that MAP and some of the other sequence-based peptides seemingly enter into cells via a nonendocytotic pathway. 11,12,16 The sequence-based peptides resulted from the fusion of a hydrophobic peptide (eg, a signal sequence, a fusion peptide) with the NLS motif, whereas the MAP peptide is a complete canonical amphipathic helix. As observed with pAntp [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] , the unconventional internalization of these peptides seems to be neither saturable nor dependent on the cell type 8,10 and could be related to their lipid-binding capacity.…”