Different mechanisms for cellular internalization of the HIV‐1 Tat‐derived cell penetrating peptide and recombinant proteins
fused to Tat

Abstract

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. A promising strategy to overcome this problem consists in the chemical conjugation (or fusion) to cell penetrating peptides (CPP) derived from proteins able to cross the plasma membrane. A large number of dierent cargo molecules such as oligonucleotides, peptides, peptide nucleic acids, proteins or even nanoparticles have been internalized in cells by this strategy. One of these translocating peptides was derived from the HIV-1 Tat protein. The mechanisms by which CPP enter cells remain unknown. Recently, convincing biochemical and genetic ®ndings has established that the full-length Tat protein was internalized in cells via the ubiquitous heparan sulfate (HS) proteoglycans. We demonstrate here that the short Tat CPP is taken up by a route that does not involve the HS proteoglycans.

Intracellular trafficking of nonviral vectors

“…Recent studies have indicated that the mode of cell entry is endocytic, 110 likely via the binding to and internalization of cell surface proteoglycans. [111][112][113] In agreement, cell entry of a nonaarginine peptide (R9) was found to be proteoglycan-mediated, as R9 was not taken up by proteoglycan-deficient CHO cells, and cell binding and entry was also inhibited by heparin. 114 Several studies have identified macropinocytosis as being the major cell entry route of TAT PTD peptides, and similar proteins, that bind proteoglycans.…”

“…118 While wild-type phage do not typically infect mammalian cells, it is possible that the context of the phage may influence entry into caveolae, although it is shown elsewhere that TAT fusion proteins alone also use caveolae to enter HeLa cells. 119 Using FACS analysis of live cells, Silhol et al 112 showed that the TAT basic domain entered proteoglycan-deficient CHO cells though the actual route of uptake was not identified. Transfection of A549 cells by lipoplexes (DOTAP or Lipofectin) decorated with TAT PTD, used here to presumably facilitate cytosolic entry, was inhibited by 41C but enhanced by chloroquine, 120 suggesting that an endocytic, possibly clathrin-mediated, pathway enabled uptake.…”

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

“…Cellular uptake was increased remarkably at 4 8C, as observed in both mammalian and plant cells (3,6,44). This may be due to low temperature-induced change in the physicochemical properties of the cell membrane, stabilizing inverted phospholipids micelles thereby favoring a higher cellular uptake of the peptides studied (45)(46)(47). Higher uptake at low temperatures suggests that CPPs transduction does not involve endocytosis but direct translocation across the cell membrane.…”

Pathway for Polyarginine Entry into Mammalian Cells

“…In the presence of a lipid bilayer, penetratin appears to form an amphipathic α-helix, which facilitates its insertion into the bilayer (44,45). HIV TAT peptide has likewise be found in a helical structure by NMR spectroscopy, suggesting that HIV TAT and perhaps other PTDs could act in a manner similar to penetratin (46).…”