Direct Visualization by Confocal Fluorescent Microscopy of the Permeation of Myristoylated Peptides Through the Cell Membrane

Abstract

SummaryTo study the permeability through the cellular membrane of synthetic peptides containing an hydrofobic moiety, we used a 13-mer myristoylated peptide labeled with a N-terminal fluorescent probe.

Investigation of the sequence and length dependence for cell-penetrating prenylated peptides

“…This allows them to avoid endosomal entrapment. Finally, we note that modification of peptides with other hydrophobic moieties such as decyl9 or myristoyl groups16 has been shown to increase a peptide’s cell-penetrating ability; like geranylgeranylation, the hydrophobicity of these modifications may impart ATP-independent uptake. The size, uptake efficiency, and internalization mechanism make prenylated cell-penetrating molecules interesting candidates for further study as cellular transport vehicles.…”

Myristoyl-Based Transport of Peptides into Living Cells

“…In the cell, the enzyme N-myristoyl transferase catalyzes the covalent attachment of myristate to the N-terminal glycine residue of a number of proteins (16,17). Myristoylation of an exogenous peptide or protein may also lead to membrane targeting (18). Surprisingly, unlike the CPPs, the cellular uptake of myristoylated cargos has received very little study.…”

“…A number of physicochemical studies have been performed on the behavior of myristoylated peptide insertion in lipid bilayers (27)(28)(29)(30)(31)(32)(33), while little has been published on the cellular uptake of such peptides (18). The in vitro studies in lipid bilayers and reconstituted cellular membranes suggest that the conjugated peptide inserts into the membrane by virtue of the myristoyl tag (28)(29)(30)(31).…”

“…Nevertheless, the molecular mechanism underlying myristoyl-based transport of peptide cargos into living cells has yet to be elucidated. The solitary mechanistic study of cellular uptake was performed by EnsenatWaser et al (18). These investigators employed confocal microscopy and a fluorescently tagged 13-mer peptide containing a myristoylated N-terminus.…”

Adenovirus-Mediated Delivery and Expression of a cAMP-Dependent Protein Kinase Inhibitor Gene to BEAS-2B Epithelial Cells Abolishes the Anti-Inflammatory Effects of Rolipram, Salbutamol, and Prostaglandin E2: A Comparison with H-89

“…Despite their high selectivity for PKA over other kinases, PKIs and inhibitory peptides derived from these proteins are poorly cell permeant and are limited to studies in permeabilized cells (Harris et al, 1997;EnsenatWaser et al, 2002). Improvements in peptide permeation can be achieved by N-myristoylation (Harris et al, 1997;Ensenat-Waser et al, 2002), but this approach is compromised by incomplete uptake into the cell and the selective partitioning of N-myristoylated peptides into the plasma membrane (Harris et al, 1997). One method to circumvent these problems is to transfect cells with plasmids encoding active peptides derived from PKI␣ (Grove et al, 1987;Olsen and Uhler, 1991a).…”