Application of a Novel Analysis To Measure the Binding of the Membrane-Translocating Peptide Penetratin to Negatively Charged Liposomes

Persson, Daniel; Thorén, Per; Herner, Mattias; Lincoln, Per; Nordén, Bengt

doi:10.1021/bi026453t

Cited by 89 publications

(139 citation statements)

References 51 publications

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“…The uptake of the penetratin and the HIV-1 Tat peptide had originally been described to be insensitive to low temperature (21,22,24) and to inhibitors of endocytosis (22,25). Penetratin was also demonstrated to traverse a pure lipid bilayer (26) without forming pores (26,27). In summary, these results were consistent with the theory of a direct translocation of the cationic peptides through the plasma membrane (21,22,28).…”

supporting

confidence: 78%

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

Fischer

Köhler

Fotin‐Mleczek

et al. 2004

Journal of Biological Chemistry

271

289

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The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the transGolgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8 -10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.

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supporting

confidence: 78%

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

Fischer

Köhler

Fotin‐Mleczek

et al. 2004

Journal of Biological Chemistry

271

289

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“…Treatment with ethylenimine [42] increased the stability, but adhesion was still detectable. Use of the full-length peptide yielded an approximate affinity for copper, K d 0.5 lm, but with large deviations of the data points from the fitted line (data not shown).…”

Section: Fluorescence Spectroscopymentioning

confidence: 94%

High‐resolution NMR studies of the zinc‐binding site of the Alzheimer's amyloid β‐peptide

et al. 2006

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Metal binding to the amyloid β‐peptide is suggested to be involved in the pathogenesis of Alzheimer's disease. We used high‐resolution NMR to study zinc binding to amyloid β‐peptide 1–40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo‐form and the holo‐form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N‐relaxation and amide proton exchange experiments on amyloid β‐peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N‐terminus, similar to a previously proposed copper‐binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169–18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β‐peptide. The dissociation constant Kd of zinc for the fragment amyloid β‐peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid β‐peptide 1–40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal‐induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal‐induced structure of the peptide counteracts aggregation.

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“…The TAT protein is an 86 amino acid long protein that is released by infected cells and is an essential regulatory gene for HIV replication [8]. In 1997, Vives et al [9] found that a 11-amino acid sequence, TAT (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57), now known as the TAT peptide or TAT PTD, can not only enter cells but is more efficient than the full length protein. It was observed that the chirality of the peptide backbone has no effect on cellular uptake of TAT peptide; inverse and retro forms were able to enter cells as efficiently as the native peptide, suggesting uptake does not require a specific binding site.…”

Section: Tat As a Prototypical Example Of A Cell-penetrating Peptidementioning

confidence: 99%

Arginine‐rich cell‐penetrating peptides

et al. 2009

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a b s t r a c tArginine-rich cell-penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell-penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell-penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.

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Application of a Novel Analysis To Measure the Binding of the Membrane-Translocating Peptide Penetratin to Negatively Charged Liposomes

Cited by 89 publications

References 51 publications

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

High‐resolution NMR studies of the zinc‐binding site of the Alzheimer's amyloid β‐peptide

Arginine‐rich cell‐penetrating peptides

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