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Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation

The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransfer...

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Autores principales: Lanyon-Hogg, Thomas, Patel, Neki V., Ritzefeld, Markus, Boxall, Katherine J., Burke, Rosemary, Blagg, Julian, Magee, Anthony I., Tate, Edward W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453399/
https://www.ncbi.nlm.nih.gov/pubmed/28296537
http://dx.doi.org/10.1177/2472555216689529
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author Lanyon-Hogg, Thomas
Patel, Neki V.
Ritzefeld, Markus
Boxall, Katherine J.
Burke, Rosemary
Blagg, Julian
Magee, Anthony I.
Tate, Edward W.
author_facet Lanyon-Hogg, Thomas
Patel, Neki V.
Ritzefeld, Markus
Boxall, Katherine J.
Burke, Rosemary
Blagg, Julian
Magee, Anthony I.
Tate, Edward W.
author_sort Lanyon-Hogg, Thomas
collection PubMed
description The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC(50) values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation.
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spelling pubmed-54533992017-06-06 Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation Lanyon-Hogg, Thomas Patel, Neki V. Ritzefeld, Markus Boxall, Katherine J. Burke, Rosemary Blagg, Julian Magee, Anthony I. Tate, Edward W. SLAS Discov Original Research The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC(50) values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation. SAGE Publications 2017-01-31 2017-04 /pmc/articles/PMC5453399/ /pubmed/28296537 http://dx.doi.org/10.1177/2472555216689529 Text en © 2017 Society for Laboratory Automation and Screening http://creativecommons.org/licenses/by/3.0/ This article is distributed under the terms of the Creative Commons Attribution 3.0 License (http://www.creativecommons.org/licenses/by/3.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
spellingShingle Original Research
Lanyon-Hogg, Thomas
Patel, Neki V.
Ritzefeld, Markus
Boxall, Katherine J.
Burke, Rosemary
Blagg, Julian
Magee, Anthony I.
Tate, Edward W.
Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation
title Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation
title_full Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation
title_fullStr Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation
title_full_unstemmed Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation
title_short Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation
title_sort microfluidic mobility shift assay for real-time analysis of peptide n-palmitoylation
topic Original Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453399/
https://www.ncbi.nlm.nih.gov/pubmed/28296537
http://dx.doi.org/10.1177/2472555216689529
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