Receptor/Transporter-independent Targeting of Functional Peptides across the Plasma Membrane

Veach, Ruth Ann; Liu, Danya; Yao, Shanshan; Chen, Yiliu; Liu, Xue Yan; Downs, Sheila; Hawiger, Jacek

doi:10.1074/jbc.m311089200

Cited by 48 publications

(45 citation statements)

References 43 publications

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“…The diffusion, pore, and micelle models imply that translocation is a rapid, temperatureand energy-independent process. Consistently with these predictions substance P, R 7 W, Tat 47-57, and the signal sequence hydrophobic region peptides have been demonstrated to translocate into live cells at 4°C or in the presence of metabolic inhibitors (22,(57)(58)(59). CPP translocation across model lipid membranes also supports this notion (60,61).…”

Section: Discussionsupporting

confidence: 66%

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

Marinova

Vukojević

Surcheva

et al. 2005

Journal of Biological Chemistry

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Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

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Section: Discussionsupporting

confidence: 66%

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

Marinova

Vukojević

Surcheva

et al. 2005

Journal of Biological Chemistry

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“…NLS was fused to the signal sequence hydrophobic segment from fibroblast growth factor 4. This hydrophobic segment serves as a membrane-translocating motif (MTM), which enables peptide or protein cargoes to cross freely the plasma membrane of multiple cell types in various organs (16, 20 -24) through a receptor/ transporter-independent mechanism (25). Of equal importance, these cell-permeant peptides carrying NLS have been shown to simultaneously block the nuclear import of multiple SRTFs in the cultured Jurkat T cell line, a process that is mediated by the shuttling molecule Rch1/importin ␣/karyopherin-␣ 2 (26).…”

Section: Staphylococcal Enterotoxin B (Seb)mentioning

confidence: 99%

Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor

Liu

Robinson

et al. 2004

Journal of Biological Chemistry

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Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin ␣/karyopherin ␣ 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor ␣, interferon ␥, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses. Staphylococcal enterotoxin B (SEB)1 causes a spectrum of human diseases, including food poisoning and non-menstrual toxic shock syndrome (NMTSS) (1, 2). SEB is one of the major virulence factors regulated by a quorum-sensing mechanism in the setting of staphylococcal infections caused by antibioticresistant strains. These high-risk community-acquired infections, which may lead to NMTSS, occur with increasing frequency as compared with the greater than 2 million hospitalacquired infections recorded annually in the United States (3, 4). Strikingly, SEB induces a fatal respiratory distress syndrome in non-human primates, suggesting its potential use as a bioweapon on the battlefield or in mass civilian settings (5, 6). Potential air-borne, water-borne, and food-borne use of SEB led to its designation by the United States Centers for Disease Control as a category B agent.In terms of its mechanism of action, SEB is avidly bound by the T cell receptor V␤ chain and by major histocompatibility complex class II proteins on dendritic cells or macrophages (7-9). The resulting intercellular "synapse" generated by SEB engagement leads to excessive production of the inflammatory cytokines tumor necrosis factor ␣ (TNF␣), interferon ␥ (IFN␥), interleukin (IL)-1␤, IL-2, and IL-6. T cell-produced inflammatory cytokines contribute to massive vascular injury, organ failure, and depending on the mode of exposure potentially lethal respiratory distress syndrome or toxic shock (1, 2, 5, 6). Active immunization prior to SEB exposure and passive immunization immediately after exposure are not readily available (6). We have designed an alternative approach to antibodymediated neutralization of SEB and related toxins by targeting a common step in their intracellular signaling to the nucleus required for inflammatory cytokine gene expression.The genes that encode inflamm...

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“…Cellular import is also involved in the regulation of intracellular pathways associated with adhesion, signaling and trafficking to the nucleus. Recent studies have suggested that, in general, cell-signaling peptides translocate a functional nuclear localization sequence directly across the plasma membrane bilayers without this sequence interacting with a receptor or transporter protein in the process (5,(8)(9)(10). However, there are no high-resolution studies on the secondary structure, folding, and membrane-interaction of cell-signaling peptides to understand their transport mechanism.…”

mentioning

confidence: 99%

Structure, Topology, and Tilt of Cell-Signaling Peptides Containing Nuclear Localization Sequences in Membrane Bilayers Determined by Solid-State NMR and Molecular Dynamics Simulation Studies

et al. 2007

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Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates through the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membraneinteraction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15±3° in POPC, while in DMPC 25±3° and 30±3° tilts were observed for SA and SKP peptides respectively. These results are in good agreement with molecular dynamics simulations, which predicts a tilt angle of 13.3° (SA in POPC), 16.4° (SKP in POPC), 22.3° (SA in DMPC) and 31.7°( SKP in POPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in the bilayer thickness without changing the phase, order, and structure of the lipid bilayers. KeywordsCell-permeable peptides; NLS; PISA wheel; tilt; hydrophobic mismatch; transmembrane helix Noninvasive delivery of peptides, proteins, oligonucleotides and other functional cargo molecules into living cells is highly dependent on their efficient transport across the plasma membrane barrier (1-7). Hydrophobic peptides have been successfully used to transport nuclear localization sequences (NLS) in order to probe intracellular signaling, which involved

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Receptor/Transporter-independent Targeting of Functional Peptides across the Plasma Membrane

Cited by 48 publications

References 43 publications

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor

Structure, Topology, and Tilt of Cell-Signaling Peptides Containing Nuclear Localization Sequences in Membrane Bilayers Determined by Solid-State NMR and Molecular Dynamics Simulation Studies

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