Cellular Uptake of Unconjugated TAT Peptide Involves Clathrin-dependent Endocytosis and Heparan Sulfate Receptors

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 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. In contrast, the caveolindependent 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.Recent advances in the identification of new molecular therapy targets and disease-relevant proteins, accelerated by the completion of the human genome project, emphasized an importance of high molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA, and small interfering RNA, for molecular therapy. However, the delivery of proteins and nucleic acids into cells is greatly hampered by the low permeability of the cell plasma membrane to polar molecules. Not surprisingly, the discovery that a number of cationic peptides known as protein transduction domains (PTDs) 1 can facilitate cytoplasmic and nuclear delivery of a conjugated cargo has attracted a lot of interest (1-3). Up to date, a wide range of cargo molecules, including low molecular weight drugs (4), oligonucleotides (5), peptides (6) and even full-length proteins (7-10), have been successfully delivered into cells using PTDs and, most importantly, the functional activity of the delivered cargo has been observed (7-10).Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Until recently, it was widely assumed that the internalization of cationic PTDs is an energy-and receptor-independent process based on direct transport through the lipid bilayer (11-15). On the other hand, 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). Moreover, the validity of some of the important data, supporting a direct transport model for synthetic TAT peptide, has been questioned in several recent ...

Arginine‐rich cell‐penetrating peptides

“…Both TAT-rhodamine [45] and TAT-GFP [41] complexes have also been shown to colocalize with cholera toxin, which is known to proceed through a caveolin-dependent pathway. However, nystatin, a compound known to inhibit caveolae formation, and filipin III had little effect on the uptake of fluorescently labeled TAT into HeLa cells or CHO cells [33].…”

“…FACS analysis following trypsin treatment indicated a relatively slow rate of uptake, comparable to that of classical markers of endocytosis. Since then, many other studies have also observed inhibition of cellular uptake at 4°C and with chemical means that induce energy depletion, indicating an energy-dependent process as the major route for the internalization of cell-penetrating peptides [27][28][29][30][31][32][33].…”

Self-Assembled Quantum Dot−Peptide Bioconjugates for Selective Intracellular Delivery

“…The classical or core CPP motif is derived from the HIV-1 Tat peptide and usually consists of 7-11 consecutive repeats of positively charged arginine residues (19). While the mechanism of CPP-mediated membrane translocation remains under investigation, increasing evidence points to the involvement of several types of endocytosis (20)(21)(22)(23)(24)(25).…”

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy

“…11,12 To characterize the role of HSPGs in TAT-mediated enhancement on cell destruction, GCV sensitivity assays were carried out in CHO-cells as well as in CHO mutant cell lines, which do not produce HSPG (pgsD-677) or produce minimal amount of glycosaminoglycans compared with wild-type cells (pgsB-618). The cell populations with 2.5, 5, 10 and 20% of TAT-TK-GFP-or TK-GFP-positive cells were exposed to GCV for 5 days.…”

“…It is proposed that the initial step for TAT PTD internalization is interaction with cell-surface heparan sulfate proteoglycans (HSPG). 11,12 Thereupon, peptide is translocated through plasma membrane, which is suggested to take place in a non-endocytic manner, such as direct penetration 13 or through different forms of endocytosis, including clathrin-mediated endocytosis, 12 caveolae-mediated endocytosis 14 or macropinocytosis. 15 Various and controversial data, however, suggest that entry of the TAT peptide can occur via more than one route and is abundantly depending on the nature of the cargo and furthermore on the target cell type.…”