Myristoyl-Based Transport of Peptides into Living Cells

Abstract

Translocation of membrane impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to peptide uptake using a cell penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid reaching a maximum within 30 minutes. Cellular uptake of the peptide lagged the membrane association, but occurred within a similar time frame. Experiments performed at 37°C vs. 4°C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 μM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cellbased biochemical studies.

Migfilin, a Molecular Switch in Regulation of Integrin Activation

“…S5, a punctate and a uniform fluorescent pattern. Similar patterns have been described in the literature of cells in which modified peptides have been taken up by cells (50,51). Similar micrographs were also obtained with A5 cells stably expressing ␣IIb␤3 (not shown).…”

S32826, A Nanomolar Inhibitor of Autotaxin: Discovery, Synthesis and Applications as a Pharmacological Tool

“…It is worth stressing that random screening of a library of diverse compounds led to the discovery of several compounds bearing similar features, S32826 being the most potent. Conversely, myristoylation is a post-translational modification known to address the targeted proteins (Boutin, 1997) as well as small peptides (Nelson et al, 2007) to plasma membranes. Nevertheless, one can also point out that the lyso-phospholipid-like compounds that were synthesized and characterized in the literature as autotaxin inhibitors have the same defaults and were at least a thousand times less potent.…”

Toll-like Receptors in the Vascular System: Sensing the Dangers Within

“…The delivery of TLR inhibitors as purified proteins has been suggested but the cost of this approach is currently prohibitory. Cell-penetrating moieties, including short cationic peptides and fatty acids, have been successfully added to inhibitory peptides to cross the cell membrane and target TLR signaling (Nelson et al, 2007;Avbelj et al, 2011). The use of microRNAs and inhibitors of ubiquitination to suppress excessive immune system activation and inflammation may be promising, but safety and efficacy as well as specificity have not yet been addressed.…”