Interaction of the third helix of Antennapedia homeodomain and a phospholipid monolayer, studied by ellipsometry and PM-IRRAS at the air–water interface

The pestivirus envelope glycoprotein E rns has RNase activity and therefore was suspected to enter cells to cleave RNA. The protein contains an RNase domain with a C-terminal extension, which shows homology with a membrane-active peptide. The modular architecture and the C-terminal homology suggested that the C terminus could be responsible for the presumed translocation. Peptides corresponding to the C-terminal domain of E rns and also the homologous L3 loop of ribotoxin II were indeed able to translocate across the eukaryotic cell membrane and were targeted to the nucleoli. The entire E rns protein was also able to translocate into the cell. Furthermore, other labeled proteins and even active enzymes could be transported inside the cell when they were attached to the C-terminal E rns peptide. Translocation was energy-independent and not mediated by a protein receptor. The peptides showed no specificity for cell type or species.

Section: Discussionsupporting

confidence: 63%

Translocation Activity of C-terminal Domain of Pestivirus Erns and Ribotoxin L3 Loop

Langedijk

2002

“…These contacts draw the PTDs in close to the plasma membrane, where, by an unknown mechanism, a rapid nonendocytic process occurs, which delivers the PTDs and their cargoes into the cell. This second step may be mediated by interactions of charged heads of phospholipids groups with the cationic residues of the PTDs, as suggested by studies from the Antennapedia PTD (18,29,(31)(32)(33)(34)(35)(36)(37). These data are supported by the fact that longer PTDs (10-and 12-mers) are better able to mediate transduction in GAG-deficient lines than short PTDs (Fig.…”

Section: Discussionmentioning

confidence: 76%

“…Internalization of Antennapedia, TAT, and arginine homopolymers has been shown to be nonsaturable and achiral, and it is not reliant on particular secondary structural elements within the PTD, as evidenced by mutagenesis experiments, use of retro-inverso peptides, ␤-peptides, peptoids, and substitution with D-enantiomers of PTD residues (19, 21, 26 -30). The broad range of transducible cell targets points toward the involvement of ubiquitously shared cellular structures, such as plasma membrane phospholipids (18,29,(31)(32)(33)(34)(35)(36)(37). Data have supported the role of heparan sulfate proteoglycans as a surface binding target for the TAT protein (38 -40), although more recent work has suggested that they are not required for internalization of the TAT PTD (23,41,42).…”

mentioning

confidence: 99%

Efficiency of Protein Transduction Is Cell Type-dependent and Is Enhanced by Dextran Sulfate

Jeffrey¹,

Shen

Watkins³

et al. 2002

142

124

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly utilized to deliver biologically active agents to a variety of cell types in vitro and in vivo. We report that in addition to previously characterized arginine-rich PTDs, including TAT, lysine homopolymers were able to mediate transduction of a wide variety of cell types, as measured by flow cytometric and enzymatic assays. The efficiency of PTD-mediated transduction was influenced by the cell type tested, although polylysine homopolymers demonstrate levels of internalization that consistently exceeded those of TAT and arginine homopolymers. Transduction of arginine/lysine-rich PTDs occurred at 4°C and following depletion of cellular ATP pools, albeit generally at reduced levels. Although transduction was reduced in Chinese hamster ovary mutant lines deficient in either heparan sulfate or glycosaminoglycan synthesis, uptake was restored to wild-type levels by incubating target cells with dextran sulfate. The enhancement of transduction by dextran sulfate suggests that electrostatic interactions play an important first step in the process by which PTDs and their cargoes traverse the plasma membrane.

“…Following fluorescently labeled Antennapedia peptide by microscopy suggested that only peptide interaction with membrane lipids is required and no pore formation occurs (14). Many studies have shown that electrostatic interactions of TAT and other PTDs with the negative charges of the membrane are important for transduction (9,(15)(16)(17). Even though TAT interacts with negatively charged membrane phospholipids (18), 2 it has also been shown that TAT has a higher affinity for negatively charged membrane sugars, such as glycosaminoglycans (18).…”

mentioning

confidence: 99%

Transactivator of Transcription Fusion Protein Transduction Causes Membrane Inversion

Moore

Payne

2004

The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT⅐GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction. Protein transduction domains (PTDs)1 have been shown to transfer a wide range of cargos across the cell membrane, including large proteins (1), polyanionic oligonucleotides (2), liposomes (3,4), and even metallic beads (5, 6). The exact mechanism that these short, positively charged PTDs use to transport such diverse molecules into cells has been the subject of much recent investigation. The process is not receptor-mediated, but there have been conflicting reports about whether transduction is temperature-dependent (7) or -independent (8 -10). Likewise, different groups have found TAT movement to be independent of endocytosis and caveolae formation (11,12), whereas others have shown that transduction is blocked by inhibitors of endocytosis (7,10). Treatment of cells with drugs that inhibit cellular transport, such as brefeldin A (inhibits Golgi transport), or metabolic processes, such as rotenone (inhibits mitochondrial respiratory chain), have also been shown to have no effect on transduction of PTDs (11, 13). Torchilin et al. (4) confirmed that neither the respiratory chain nor the cytoskeleton is involved by showing that uptake of TAT attached to liposomes occurred at low temperatures and in the presence of sodium azide or iodoacetamine, respectively. Following fluorescently labeled Antennapedia peptide by microscopy suggested that only peptide interaction with membrane lipids is required and no pore formation occurs (14). Many studies have shown that electrostatic interactions of TAT and other PTDs with the negative charges of the membrane are important for transduction (9,(15)(16)(17). Even though TAT interacts with negatively charged membrane phospholipids (18), 2 it has also been shown that TAT has a higher affinity for negatively charged membrane sugars, such as glycosaminoglycans (18). Recent data concerning TAT fusion protein transduction suggest that PTDs may use lipid rafts to get into cells (7,1...