Cellular Uptake Studies with -Peptides

Rueping, Magnus; Mahajan, Yogesh R.; Sauer, Markus; Seebàch, Dieter

doi:10.1002/1439-7633(20020301)3:2/3<257::aid-cbic257>3.0.co;2-s

Cited by 148 publications

(108 citation statements)

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“…Early mechanistic studies on the cellular entry of PTDs were performed with fixed cells (11,13,(22)(23)(24)(25)(26)(27)(28). Fixation is believed to permeate membranes (30), and allows vesicle-entrapped peptides and proteins to travel to new locations (29,30,32).…”

Section: Discussionmentioning

confidence: 99%

“…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).Early mechanistic studies on the cellular entry of PTDs were performed with fixed cells (11,13,(22)(23)(24)(25)(26)(27)(28). Fixation is believed to permeate membranes (30), and allows vesicle-entrapped peptides and proteins to travel to new locations (29,30,32).…”

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Pathway for Polyarginine Entry into Mammalian Cells

2004

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Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R 9 ), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R 9 is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Co-localization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R 9 to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R 9 for heparin (K d = 109 nM). Finally, R 9 is shown to promote the leakage of liposomes, but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves: (1) binding to cell-surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles. Facilitating the delivery of molecules into mammalian cells is of substantial interest (1-6).Developing the means to do so can generate both insights into cellular function and potential applications in biomedicine. As early as 1965, Ryser noted that mixing polylysine or other cationic macromolecules with proteins greatly increases the uptake of those proteins by cells (7). In 1988, Frankel and Green independently discovered that the RNA-binding HIV 1 TAT protein was capable of crossing lipid bilayers (8,9). The dissection of HIV TAT function has revealed that a small cationic domain, residues 47-57, is responsible for transduction (10). Other cationic, nucleic acid-binding sequences are likewise capable of entering cells (11,12). Peptide-like molecules and even non-peptidyl polycations are capable of crossing lipid bilayers (11,13). These cationic "protein transduction domains" (PTDs) are not only capable of entering cells but also can be used to deliver molecular cargo (14-16), such as proteins, oligonucleotides, and small molecules (1,(17)(18)(19)(20).What are the physicochemical characteristics of cationic PTDs? A natural backbone is not necessary for transduction, as D-amino acid (21), peptoid (18), oligocarbamate (22), β-peptide (23,24), and 6-aminocaproic acid (25) analogs are capable of entering cells. In contrast, the positive charge of cationic PTD is essential for transduction, and arginine is preferred over † This work was supported by National Institutes of Health Grant GM44783. 1 Abbreviations: CHO, Chinese hamster ovary; CS, condroitin sulfate; GAG, glycosaminoglycan; HIV, human immunodeficiency virus; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; PC, phosphatidyl choline; PTD, protein transduction domain; R 9 , nonaarginine; TAT, transactivator of transcription; TAMRA-R 9 , 5-(and 6-)carboxytetramethylrhodamine conjugat...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Pathway for Polyarginine Entry into Mammalian Cells

2004

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“…The studies reported here rep- 2 An attempt was made to conduct live cell studies as part of the previous study (10), but subsequent experiments have revealed that the conditions employed led to rapid cell death. Rueping et al (11) have reported cell entry by a ␤-homoarginine oligomer, and García-Echeverría and Ruetz (12) have reported entry by ␤-homolysine oligomers. 4 into the cytoplasm and nucleus of cells.…”

Section: Discussionmentioning

confidence: 99%

Cytoplasmic and Nuclear Delivery of a TAT-derived Peptide and a β-Peptide after Endocytic Uptake into HeLa Cells

Potocky¹,

Menon

Gellman³

2003

Journal of Biological Chemistry

217

207

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Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural ␤-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47-57), as well as a similarly labeled 12-residue ␤-peptide, ␤-(VRR) 4 , in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47-57)) or nucleolus (␤-(VRR) 4 ). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47-57) and ␤-(VRR) 4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.There is widespread interest in the use of protein-derived peptides (1-3), designed peptides (4 -7), and oligomers constructed from unnatural building blocks (8 -12) as vectors to deliver molecular "cargo" to targets within the cytoplasm and nucleus of eukaryotic cells (13-21). Delivery requires translocation of the peptide across the plasma membrane or an intracellular membrane of a living cell, a behavior that has been ascribed to arginine-rich peptides and related oligomers (22,23). The prototypical sequences that constitute these so-called protein transduction domains are derived from the human immunodeficiency virus (HIV) 1 transactivator of transcription (TAT) protein, the antennapedia (Antp) homeodomain, and the herpes simplex virus (HSV) type 1 DNA-binding protein VP22, all of which are rich in arginine residues (13,15,16). The mechanism by which these highly charged peptides are able to translocate from the extracellular milieu into the cytoplasm and nucleus without breach of the plasma membrane permeability barrier is unknown. One possible translocation mechanism is direct transfer of the peptide across the plasma membrane by means of a reverse micelle, ...

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“…Some ␤-peptides possess antimicrobial activities (4,5,25,38), bind to the human somatostatin receptor (12,21), function as inhibitors of human immunodeficiency virus type 1 replication (39), and inhibit p53-hDM2 interaction (18). Cationic ␤-peptides cross bacterial and mammalian cell membranes and can be considered a new group of cell-penetrating peptides (13,26,31). Because they are able to form different helices (11) and to mimic amphipathic ␣-peptidic helices and turns (7,41), ␤-peptides have a high potential for being developed as novel pharmaceutically active substances that function as stable peptidomimetics (21,30,34).…”

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A Novel β-Peptidyl Aminopeptidase (BapA) from Strain 3-2W4 Cleaves Peptide Bonds of Synthetic β-Tri- and β-Dipeptides

et al. 2005

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A novel bacterial strain that was capable of growing on the ␤-tripeptide H-␤hVal-␤hAla-␤hLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the ␤-tripeptide and the ␤-dipeptide H-␤hAla-␤hLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated ␤-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The ␤-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a ␤-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily.

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Cellular Uptake Studies with -Peptides

Cited by 148 publications

References 7 publications

Pathway for Polyarginine Entry into Mammalian Cells

Pathway for Polyarginine Entry into Mammalian Cells

Cytoplasmic and Nuclear Delivery of a TAT-derived Peptide and a β-Peptide after Endocytic Uptake into HeLa Cells

A Novel β-Peptidyl Aminopeptidase (BapA) from Strain 3-2W4 Cleaves Peptide Bonds of Synthetic β-Tri- and β-Dipeptides

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