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

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 transloc...

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Autores principales:	Evgeniya Trofimenko, Gianvito Grasso, Mathieu Heulot, Nadja Chevalier, Marco A Deriu, Gilles Dubuis, Yoan Arribat, Marc Serulla, Sebastien Michel, Gil Vantomme, Florine Ory, Linh Chi Dam, Julien Puyal, Francesca Amati, Anita Lüthi, Andrea Danani, Christian Widmann
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	cell-penetrating peptides water pores potassium channels membrane potential TAT In silico modeling Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/98b234728cca409a8a4e7ab3072baed6
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spelling	oai:doaj.org-article:98b234728cca409a8a4e7ab3072baed62021-12-02T16:24:18ZGenetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore10.7554/eLife.698322050-084Xe69832https://doaj.org/article/98b234728cca409a8a4e7ab3072baed62021-10-01T00:00:00Zhttps://elifesciences.org/articles/69832https://doaj.org/toc/2050-084XCell-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 CPP 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 membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.Evgeniya TrofimenkoGianvito GrassoMathieu HeulotNadja ChevalierMarco A DeriuGilles DubuisYoan ArribatMarc SerullaSebastien MichelGil VantommeFlorine OryLinh Chi DamJulien PuyalFrancesca AmatiAnita LüthiAndrea DananiChristian WidmanneLife Sciences Publications Ltdarticlecell-penetrating peptideswater porespotassium channelsmembrane potentialTATIn silico modelingMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cell-penetrating peptides water pores potassium channels membrane potential TAT In silico modeling Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	cell-penetrating peptides water pores potassium channels membrane potential TAT In silico modeling Medicine R Science Q Biology (General) QH301-705.5 Evgeniya Trofimenko Gianvito Grasso Mathieu Heulot Nadja Chevalier Marco A Deriu Gilles Dubuis Yoan Arribat Marc Serulla Sebastien Michel Gil Vantomme Florine Ory Linh Chi Dam Julien Puyal Francesca Amati Anita Lüthi Andrea Danani Christian Widmann Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
description	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 CPP 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 membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.
format	article
author	Evgeniya Trofimenko Gianvito Grasso Mathieu Heulot Nadja Chevalier Marco A Deriu Gilles Dubuis Yoan Arribat Marc Serulla Sebastien Michel Gil Vantomme Florine Ory Linh Chi Dam Julien Puyal Francesca Amati Anita Lüthi Andrea Danani Christian Widmann
author_facet	Evgeniya Trofimenko Gianvito Grasso Mathieu Heulot Nadja Chevalier Marco A Deriu Gilles Dubuis Yoan Arribat Marc Serulla Sebastien Michel Gil Vantomme Florine Ory Linh Chi Dam Julien Puyal Francesca Amati Anita Lüthi Andrea Danani Christian Widmann
author_sort	Evgeniya Trofimenko
title	Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
title_short	Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
title_full	Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
title_fullStr	Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
title_full_unstemmed	Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
title_sort	genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/98b234728cca409a8a4e7ab3072baed6
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Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

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