Abstract
At concentrations exceeding 10 μM, arginine-rich cell-penetrating peptides (CPPs) trigger a rapid cytoplasmic import that involves activation of acid sphingomyelinase (ASMase). ASMase activation occurs through a variety of stress signals and has also been related to the reorganization of membrane microdomains during entry of pathogens. However, in none of these cases has the initial trigger for ASMase activation been established on a molecular level. We here show that rapid cytosolic CPP import depends upon an increase in intracellular calcium, likely caused by modulation of the Orai1 calcium channel. At low peptide concentration, cytoplasmic import could be induced by thapsigargin, a known activator of Orai1. Compounds known to block Orai1 inhibited rapid uptake. Peptide-mediated modulation of Orai1 involved cell surface sialic acids as inhibition of sialylation as well as chemical blocking of sialic acids reduced rapid cytoplasmic uptake, which could be reconstituted by thapsigargin. These results establish a link between the known propensity of arginine-rich CPPs to interact with the glycocalyx and calcium influx as the initial step triggering direct cytosolic peptide uptake.
Highlights
Cell-penetrating peptides (CPPs) induce the efficient cellular uptake ofmolecules that by themselves enter cells only inefficiently
In an effort to elucidate the underlying molecular mechanism of this import pathway, we had demonstrated an involvement of acid sphingomyelinase, which converts sphingomyelin into ceramide [5]
This mechanism would imply an increase in intracellular calcium in the uptake of R9
Summary
Cell-penetrating peptides (CPPs) induce the efficient cellular uptake of (macro)molecules that by themselves enter cells only inefficiently. At low concentrations of nona-L-arginine (R9) (2–5 μM), the predominant uptake mechanism that is observed in HeLa and other cells is endocytosis. Uptake at higher concentrations (10–20 μM) occurs by direct cytoplasmic entry through localized areas of the plasma membrane which we had coined nucleation zones [3] (for a review: [4]). Several other groups have described this process of direct uptake in various adherent as well as non-adherent cell types for different types of arginine-rich CPPs [6,7,8]. Direct uptake of peptide into the cytosol bypasses endosomal sequestration and is the preferred mode of entry in drug delivery. As such, unraveling the molecular mechanism by which arginine-rich CPPs induce direct cytosolic uptake is warranted
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