Abstract
Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.
Highlights
Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential
We studied the effects of ATP depletion and low temperature on TAT peptide uptake by human peripheral blood mononuclear macrophages and HUVEC endothelial cells
These recent findings have led to a major reevaluation of key characteristics of PTD internalization and the role of endocytosis in PTD-mediated uptake
Summary
Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. We demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATP- and temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. There have been indications that uptake of full-length TAT protein, from which one of the most commonly used PTDs referred to as TAT peptide is derived, occurs via endocytosis and depends on cell surface heparan sulfate receptors [16]. As in the case of the stable cell lines used in our earlier work, TAT entry into several different primary cells is found to be ATP- and temperature-dependent, indicating the involvement of endocytosis. A significant part of TAT peptide uptake involves heparan sulfate receptors, these receptors are not a prerequisite for TAT entry
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