Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

Abstract

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.

Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation

“…although not yet associated with specific functions (Marinova et al, 2005;Oehlke et al, 2005) while others have revealed a receptor-mediated accumulation of intact peptide ligands into cells/tissues (e.g., Angiotensin II, BK; van Kats et al, 1997;Lamb et al, 2001;Li et al, 2006).…”

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

“…Interacting with the lipid phase peptides may translocate through membrane by forming pores or inverted micelles or diffuse through the bilayer (11). Peptide diffusion has been considered as improbable because it requires high desolvation energy (53,54). Another model proposes that hydrophilic CPPs translocate across the membrane by forming pores through oligomerization of several peptide molecules.…”

Interactions of KLA Amphipathic Model Peptides with Lipid Monolayers

“…It translocates across the cellular membrane without disrupting the latter's integrity and has been shown to deliver various cargos into cells. [13] KLAL has five positively charged lysine side chains and first binds to negatively charged membranes through electrostatic interactions before perturbing the membrane. The KLAL-induced release of fluorescent dyes from vesicles, as well as the peptide's antibacterial and haemolytic activities, have been investigated in detail.…”

“…[11,12] Specifically, lysine-rich model peptides have been shown to deliver various cargos into cells. [13] One example is the cationic 18-mer KLAL (KLALKLALKALKAALKLA-NH 2 ), synthesised together with its complete double d-amino acid replacement set as a model compound for forming amphipathic helices and analysing lipid-peptide interactions. [14,15] The peptide is highly active towards bacteria and lyses red blood cells; however, at nonlytic concentrations, it acts as a cell-penetrating peptide (CPP).…”