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

  1. Evgeniya Trofimenko
  2. Gianvito Grasso
  3. Mathieu Heulot
  4. Nadja Chevalier
  5. Marco A Deriu
  6. Gilles Dubuis
  7. Yoan Arribat
  8. Marc Serulla
  9. Sebastien Michel
  10. Gil Vantomme
  11. Florine Ory
  12. Lihn Chi Dam
  13. Julien Puyal
  14. Francesca Amati
  15. Anita Lüthi
  16. Andrea Danani
  17. Christian Widmann  Is a corresponding author
  1. University of Lausanne, Switzerland
  2. Università della Svizzera italiana, Scuola Universitaria Professionale della Svizzera Italiana, Switzerland
  3. Politecnico di Torino, Italy
  4. Université de Lausanne, Switzerland

Abstract

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2(-5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPPs cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Data availability

DNA sequencing data from the CRISPR/Cas9-based screens are available through the following link: https://www.ncbi.nlm.nih.gov/sra/SRP161445.

The following data sets were generated

Article and author information

Author details

  1. Evgeniya Trofimenko

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4910-5324
  2. Gianvito Grasso

    Dalle Molle Institute for Artificial Intelligence Research, Università della Svizzera italiana, Scuola Universitaria Professionale della Svizzera Italiana, Ticino, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Mathieu Heulot

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  4. Nadja Chevalier

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Marco A Deriu

    Bioingegneria Industriale, Politecnico di Torino, Torino, Italy
    Competing interests
    The authors declare that no competing interests exist.
  6. Gilles Dubuis

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Yoan Arribat

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0952-5279
  8. Marc Serulla

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4166-1556
  9. Sebastien Michel

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  10. Gil Vantomme

    Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7441-0737
  11. Florine Ory

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  12. Lihn Chi Dam

    Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  13. Julien Puyal

    Department of Fundamental Neurology, Université de Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  14. Francesca Amati

    Department of Physiology, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  15. Anita Lüthi

    Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4954-4143
  16. Andrea Danani

    Dalle Molle Institute for Artificial Intelligence Research, Università della Svizzera italiana, Scuola Universitaria Professionale della Svizzera Italiana, Ticino, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  17. Christian Widmann

    Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
    For correspondence
    Christian.Widmann@unil.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6881-0363

Funding

Swiss National Science Foundation (CRSII3_154420,IZCSZ0-174639)

  • Christian Widmann

Swiss National Science Foundation (158116)

  • Nadja Chevalier

Swiss National Science Foundation (320030_170062)

  • Francesca Amati

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All experiments were performed according to the principles of laboratory animal care and Swiss legislation under ethical approval (Swiss Animal Protection Ordinance; permit number VD3374.a).

Reviewing Editor

  1. Patricia Bassereau, Institut Curie, France

Version history

  1. Preprint posted: February 26, 2020 (view preprint)
  2. Received: April 27, 2021
  3. Accepted: October 28, 2021
  4. Accepted Manuscript published: October 29, 2021 (version 1)
  5. Version of Record published: December 2, 2021 (version 2)

Copyright

© 2021, Trofimenko et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Evgeniya Trofimenko
  2. Gianvito Grasso
  3. Mathieu Heulot
  4. Nadja Chevalier
  5. Marco A Deriu
  6. Gilles Dubuis
  7. Yoan Arribat
  8. Marc Serulla
  9. Sebastien Michel
  10. Gil Vantomme
  11. Florine Ory
  12. Lihn Chi Dam
  13. Julien Puyal
  14. Francesca Amati
  15. Anita Lüthi
  16. Andrea Danani
  17. Christian Widmann
(2021)
Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
eLife 10:e69832.
https://doi.org/10.7554/eLife.69832

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https://doi.org/10.7554/eLife.69832

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