Cell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis and have capacity to deliver macromolecular cargo into cells. Although the involvement of various endocytic routes has been described it is still unclear which interactions are involved in eliciting an uptake response and to what extent affinity for particular cell surface components may determine the efficiency of a particular CPP. Previous biophysical studies of the interaction between CPPs and either lipid vesicles or soluble sugar-mimics of cell surface proteoglycans, the two most commonly suggested CPP binding targets, have not allowed quantitative correlations to be established. We here explore the use of plasma membrane vesicles (PMVs) derived from cultured mammalian cells as cell surface models in biophysical experiments. Further, we examine the relationship between affinity for PMVs and uptake into live cells using the CPP penetratin and two analogs enriched in arginines and lysines respectively. We show, using centrifugation to sediment PMVs, that the amount of peptide in the pellet fraction correlates linearly with the degree of cell internalization and that the relative efficiency of all-arginine and all-lysine variants of penetratin can be ascribed to their respective cell surface affinities. Our data show differences between arginine- and lysine-rich variants of penetratin that has not been previously accounted for in studies using lipid vesicles. Our data also indicate greater differences in binding affinity to PMVs than to heparin, a commonly used cell surface proteoglycan mimic. Taken together, this suggests that the cell surface interactions of CPPs are dependent on several cell surface moieties and their molecular organization on the plasma membrane.
TRPV1 is a tetrameric voltage-sensitive cation channel that is activated by heat and vanilloids. The architecture of TRP channels are thought to be related to Kv channels, with each subunit containing six transmembrane segments. In addition to being activated by capsaicin, TRPV1 is activated by Double-Knot Toxin (DkTx), a protein toxin purified from the Chinese Bird Spider, Selenocosmia huwena (Bohlen et al. 2010, Cell 141, 834-35). DkTx is unique in that it contains two Inhibitor Cysteine Knots (ICK) motifs connected by a peptide linker. Although these ICK motifs are related to those found in tarantula toxins that target voltage sensors in Kv channels, DkTx does not appear to interact with classical voltage-activated cation channels. We set out to explore the mechanism of DkTx activation of TRPV1, and began by producing the toxin in E. Coli and testing for activity against TRPV1. DkTx was expressed as a fusion with bacterial Ketosteroid Isomerase (KSI), cleaved from KSI using hydroxylamine, and purified using reverse phase HPLC. Reduced DkTx was folded in vitro in a solution containing (GSH/GSSG) and guanidine HCl, and the folding reaction monitored by HPLC. Using this procedure we obtained a predominant species of the toxin that was further purified by reverse phase HPLC. When tested for activity on TRPV1 expressed in Xenopus laevis oocytes, DkTx produced robust and slowly reversible activation of the channel when voltage clamped at À60 mV. At a concentration of 2mM, DkTx produced comparable activation to 2 mM capsaicin, suggesting that the apparent affinity of the recombinant toxin is similar to that reported for the native toxin. We are currently working to solve the structure of DkTx using NMR and further investigating it's mechanism of activation.
TRPV1 is a tetrameric voltage-sensitive cation channel that is activated by heat and vanilloids. The architecture of TRP channels are thought to be related to Kv channels, with each subunit containing six transmembrane segments. In addition to being activated by capsaicin, TRPV1 is activated by Double-Knot Toxin (DkTx), a protein toxin purified from the Chinese Bird Spider, Selenocosmia huwena (Bohlen et al. 2010, Cell 141, 834-35). DkTx is unique in that it contains two Inhibitor Cysteine Knots (ICK) motifs connected by a peptide linker. Although these ICK motifs are related to those found in tarantula toxins that target voltage sensors in Kv channels, DkTx does not appear to interact with classical voltage-activated cation channels. We set out to explore the mechanism of DkTx activation of TRPV1, and began by producing the toxin in E. Coli and testing for activity against TRPV1. DkTx was expressed as a fusion with bacterial Ketosteroid Isomerase (KSI), cleaved from KSI using hydroxylamine, and purified using reverse phase HPLC. Reduced DkTx was folded in vitro in a solution containing (GSH/GSSG) and guanidine HCl, and the folding reaction monitored by HPLC. Using this procedure we obtained a predominant species of the toxin that was further purified by reverse phase HPLC. When tested for activity on TRPV1 expressed in Xenopus laevis oocytes, DkTx produced robust and slowly reversible activation of the channel when voltage clamped at À60 mV. At a concentration of 2mM, DkTx produced comparable activation to 2 mM capsaicin, suggesting that the apparent affinity of the recombinant toxin is similar to that reported for the native toxin. We are currently working to solve the structure of DkTx using NMR and further investigating it's mechanism of activation.
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