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Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internal...

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Autores principales: Gao, Xinxin, Stanger, Karen, Kaluarachchi, Harini, Maurer, Till, Ciepla, Paulina, Chalouni, Cecile, Franke, Yvonne, Hannoush, Rami N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062073/
https://www.ncbi.nlm.nih.gov/pubmed/27734922
http://dx.doi.org/10.1038/srep35179
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author Gao, Xinxin
Stanger, Karen
Kaluarachchi, Harini
Maurer, Till
Ciepla, Paulina
Chalouni, Cecile
Franke, Yvonne
Hannoush, Rami N.
author_facet Gao, Xinxin
Stanger, Karen
Kaluarachchi, Harini
Maurer, Till
Ciepla, Paulina
Chalouni, Cecile
Franke, Yvonne
Hannoush, Rami N.
author_sort Gao, Xinxin
collection PubMed
description Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.
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spelling pubmed-50620732016-10-24 Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking Gao, Xinxin Stanger, Karen Kaluarachchi, Harini Maurer, Till Ciepla, Paulina Chalouni, Cecile Franke, Yvonne Hannoush, Rami N. Sci Rep Article Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery. Nature Publishing Group 2016-10-13 /pmc/articles/PMC5062073/ /pubmed/27734922 http://dx.doi.org/10.1038/srep35179 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Gao, Xinxin
Stanger, Karen
Kaluarachchi, Harini
Maurer, Till
Ciepla, Paulina
Chalouni, Cecile
Franke, Yvonne
Hannoush, Rami N.
Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
title Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
title_full Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
title_fullStr Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
title_full_unstemmed Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
title_short Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
title_sort cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062073/
https://www.ncbi.nlm.nih.gov/pubmed/27734922
http://dx.doi.org/10.1038/srep35179
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