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Design rules for efficient endosomal escape

The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protei...

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Autores principales: Zoltek, Madeline, Vázquez, Angel, Zhang, Xizi, Dadina, Neville, Lesiak, Lauren, Schepartz, Alanna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635116/
https://www.ncbi.nlm.nih.gov/pubmed/37961597
http://dx.doi.org/10.1101/2023.11.03.565388
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author Zoltek, Madeline
Vázquez, Angel
Zhang, Xizi
Dadina, Neville
Lesiak, Lauren
Schepartz, Alanna
author_facet Zoltek, Madeline
Vázquez, Angel
Zhang, Xizi
Dadina, Neville
Lesiak, Lauren
Schepartz, Alanna
author_sort Zoltek, Madeline
collection PubMed
description The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a single-molecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.
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spelling pubmed-106351162023-11-13 Design rules for efficient endosomal escape Zoltek, Madeline Vázquez, Angel Zhang, Xizi Dadina, Neville Lesiak, Lauren Schepartz, Alanna bioRxiv Article The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a single-molecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo. Cold Spring Harbor Laboratory 2023-11-04 /pmc/articles/PMC10635116/ /pubmed/37961597 http://dx.doi.org/10.1101/2023.11.03.565388 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Zoltek, Madeline
Vázquez, Angel
Zhang, Xizi
Dadina, Neville
Lesiak, Lauren
Schepartz, Alanna
Design rules for efficient endosomal escape
title Design rules for efficient endosomal escape
title_full Design rules for efficient endosomal escape
title_fullStr Design rules for efficient endosomal escape
title_full_unstemmed Design rules for efficient endosomal escape
title_short Design rules for efficient endosomal escape
title_sort design rules for efficient endosomal escape
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635116/
https://www.ncbi.nlm.nih.gov/pubmed/37961597
http://dx.doi.org/10.1101/2023.11.03.565388
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