Membrane potential drives direct translocation of cell-penetrating peptides

Gao, Xinli; Hong, Song; Liu, Zhi Ping; Yue, Tongtao; Dobnikar, Jure; Zhang, Xuehua

doi:10.1039/c8nr10447f

Cited by 38 publications

(43 citation statements)

References 64 publications

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“…4A-B, Movie S6-7). Previous observations reporting a role for the plasma membrane potential in CPP penetration into or through bilipidic membranes ( 50, 59–63 ) support these findings. However, note that translocation of single CPP molecules had not been observed before the present work.…”

Section: Resultssupporting

confidence: 70%

“…Specifically, asymmetric multi-component physiological membrane ( 49 ) was constructed and subjected to a constant electric field (Fig. S9A), following a widely applied computational procedure ( 50–58 ). In our simulations we have used a physiological plasma cell membrane-like composition (for both inner and outer leaflet) while earlier studies have employed simpler membrane composition ( 50, 59–63 ).…”

Section: Resultsmentioning

confidence: 99%

“…S9A), following a widely applied computational procedure ( 50–58 ). In our simulations we have used a physiological plasma cell membrane-like composition (for both inner and outer leaflet) while earlier studies have employed simpler membrane composition ( 50, 59–63 ). The application of transmembrane potential triggered the following molecular events.…”

Section: Resultsmentioning

confidence: 99%

“…We next performed Molecular Dynamics simulations in the presence or in the absence of a membrane potential generated by setting an ion imbalance ( 50, 69–71 ) to determine the behavior of CPPs and their potential ability to move across membranes (Fig. S9F).…”

Section: Resultsmentioning

confidence: 99%

“…(B) Quantitation of CPP localization in hyperpolarized or depolarized conditions based on coarse-grained molecular dynamics simulations. Membrane hyperpolarization was achieved through a net charge difference of 30 positive ions between intracellular and extracellular space in a double bilayer system ( 50, 69–71 ) obtaining a transmembrane potential of 2.2 V. (C) Quantitation of the percentage of cells with cytoplasmic staining after the indicated treatment. The indicated compounds (32 mg/ml PI, 200 mg/ml dextran, 40 mM CPP, except MAP, which was used at 20 mM) were incubated for 30 minutes on HeLa cells.…”

Section: Resultsmentioning

confidence: 99%

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Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Trofimenko

Grasso

Heulot

et al. 2020

Preprint

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30Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. CPPs 31 provide efficient methodology to transfer bioactive molecules in cells, in particular in 32 conditions when transcription or translation of cargo-encoding sequences is not 33 desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined 34 and controversial. This work identifies potassium channels as key regulators of cationic 35 CPP translocation. Using a CRISPR/Cas9-based screening, we discovered that 36 KCNQ5, KCNN4, and KCNK5 positively modulate CPP cellular direct translocation by 37 reducing transmembrane potential (Vm). Cationic CPPs further decrease the Vm to 38 megapolarization values (about -150 mV) leading to the formation of ~2 nm-wide water 39 pores used by CPPs to access the cell's cytoplasm. Pharmacological manipulation to 40 lower transmembrane potential boosted CPPs cellular uptake in zebrafish and mouse 41 models. Besides identifying the first genes that regulate CPP translocation, this work 42 characterizes key mechanistic steps used by CPPs to cross cellular membrane. This 43 opens the ground for pharmacological strategies augmenting the susceptibility of cells 44 to capture CPP-linked cargos in vitro and in vivo. 45 46Cell-penetrating peptides (CPPs) are non-toxic molecules of 5-30 amino acids that can 47 translocate into living cells. CPPs can be hooked to a variety of cargos (siRNAs, DNA, 48 polypeptides, liposomes, nanoparticles, etc.) to allow their transport into cells for 49 therapeutic or experimental purposes (1-10). The origin of CPPs is diverse. For 50 example, TAT48-57 is a 10 amino-acid fragment derived from the trans-activator of 51 transcription (TAT) HIV-1 protein (11, 12), penetratin is a 16 amino-acid peptide 52 derived from the Antennapedia Drosophila melanogaster protein (13), and MAP (model 53 amphipatic peptide) is a synthetic alanine/leucine/lysine-rich peptide (14). The vast 54 majority of CPPs are cationic (1, 3, 6, 7). 55How CPPs enter cells is debated and not fully characterized at the molecular level 56 (reviewed in (1-8)). Due to this knowledge gap, it is difficult to ameliorate CPP cellular 57 entry and this slows down development of CPP-based interventions. Two main modes 58 of CPP entry have been described: endocytosis and direct translocation(1-8). 59Endocytosed CPPs gain cytosolic access by escaping endosomes. Direct 60 translocation has been proposed to occur through transient pore formation or 61 membrane destabilization. Endocytosis and direct translocation are not mutually 62 exclusive. Several entry routes can be followed simultaneously by a given CPP in a 63 given cell line (9, 10). 64Here, we used CRISPR/Cas9-screenings to identify proteins required for the cellular 65 uptake of CPPs. This approach identified three potassium channels as mandatory for 66 the direct translocation of CPPs into various cell types. Further, we highlighted the 67 requirement of an appropriate membrane potential to generate a 2 nm-wide water 68 pores through which...

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Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Trofimenko

Grasso

Heulot

et al. 2020

Preprint

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Peptide Structure: Electrophysiological Analysis, Nuclear Magnetic Resonance Analysis, and Molecular Dynamics Simulation of Direct Penetration of Cell‐Penetrating Peptides through Bilayer Lipid Membranes

Izumi¹,

Mijiddorj²,

Saito³

et al. 2022

Cell‐Penetrating Peptides

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Kinetic analysis of cellular internalization and expulsion of unstructured D‐chirality cell penetrating peptides

et al. 2020

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Most cell penetrating peptides (CPPs) are unstructured and susceptible to proteolytic degradation. One alternative is to incorporate D‐chirality amino acids into unstructured CPPs to allow for enhanced uptake and intracellular stability. This work investigates CPP internalization using a series of time, concentration, temperature, and energy dependent studies, resulting in a three‐fold increase in uptake and 50‐fold increase in stability of D‐chirality peptides over L‐chirality counterparts. CPP internalization occurred via a combination of direct penetration and endocytosis, with a percentage of internalized CPP expelling from cells in a time‐dependent manner. Mechanistic studies identified that cells exported the intact internalized D‐chirality CPPs via an exocytosis independent pathway, analogous to a direct penetration method out of the cells. These findings highlight the potential of a D‐chirality CPP as bio‐vector in therapeutic and biosensing applications, but also identify a new expulsion method suggesting a relationship between uptake kinetics, intracellular stability, and export kinetics.

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Membrane potential drives direct translocation of cell-penetrating peptides

Abstract: We report the molecular dynamics evidence for the direct translocation of CPPs across the membrane driven by the membrane electrostatic potential.

Cited by 38 publications

References 64 publications

Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Peptide Structure: Electrophysiological Analysis, Nuclear Magnetic Resonance Analysis, and Molecular Dynamics Simulation of Direct Penetration of Cell‐Penetrating Peptides through Bilayer Lipid Membranes

Kinetic analysis of cellular internalization and expulsion of unstructured D‐chirality cell penetrating peptides

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