The prospect of altering intracellular protein function with extracellularly applied peptides or proteins has been an attractive possibility since the ability of extracellular TAT was shown to activate transcription in the early 1990’s. Here, we show that grafting arginines onto a well-structured miniature protein results in increased structure-dependent uptake with fewer positive charges. The nature of the positive charge distribution is critical, because relocating a negatively charged fluorescein away from the positively charged arginines on the C-terminal tail resulted in greater cell uptake. The improved activity of these peptides allowed us to track their cellular uptake using substantially lower concentration than has been previously reported. Using fluorescently labeled versions of these proteins, we visualzed labeled peptides in all endosomal intermediates from clathrin-coated pits to lysosomes. In dual-color experiments, arginine-bearing miniature proteins colocalized with TAT and octa-arginine, indicating that the cellular distribution of ‘canonical’ cell penetrating peptides is identical to the peptides described here and indicating that all peptides examined follow an endosomal route from the cell surface to the lysosome. Uptake is inhibited by small molecules that disrupt the function of dynamin or that block the uptake of transferrin. Cholesterol may play a role in the trafficking because extracting plasma membrane cholesterol by incubating cells with methyl-beta-cyclodextrin changes the distribution of fluorescence on the cell surface. In some cases, peptides that accumulate in lysosomes can be photodynamically redistributed to the cytoplasm. These miniature proteins thus provide a useful probe to examine the route of uptake for cell penetrating peptides into mammalian cells, as well as potential scaffolds for peptide with the ability to modulate cellular function.
This preliminary program is subject to change.
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Enhanced Cell Uptake and Differential Trafficking Revealed by Discrete Arginine Topologies Grafted Onto a Miniature Protein
The prospect of altering intracellular protein function with extracellularly applied peptides or proteins has been an attractive possibility since the ability of extracellular TAT was shown to activate transcription in the early 1990’s. Here, we show that grafting arginines onto a well-structured miniature protein results in increased structure-dependent uptake with fewer positive charges. The nature of the positive charge distribution is critical, because relocating a negatively charged fluorescein away from the positively charged arginines on the C-terminal tail resulted in greater cell uptake. The improved activity of these peptides allowed us to track their cellular uptake using substantially lower concentration than has been previously reported. Using fluorescently labeled versions of these proteins, we visualzed labeled peptides in all endosomal intermediates from clathrin-coated pits to lysosomes. In dual-color experiments, arginine-bearing miniature proteins colocalized with TAT and octa-arginine, indicating that the cellular distribution of ‘canonical’ cell penetrating peptides is identical to the peptides described here and indicating that all peptides examined follow an endosomal route from the cell surface to the lysosome. Uptake is inhibited by small molecules that disrupt the function of dynamin or that block the uptake of transferrin. Cholesterol may play a role in the trafficking because extracting plasma membrane cholesterol by incubating cells with methyl-beta-cyclodextrin changes the distribution of fluorescence on the cell surface. In some cases, peptides that accumulate in lysosomes can be photodynamically redistributed to the cytoplasm. These miniature proteins thus provide a useful probe to examine the route of uptake for cell penetrating peptides into mammalian cells, as well as potential scaffolds for peptide with the ability to modulate cellular function.