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A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these met...

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Autores principales: Ehrnhoefer, Dagmar E., Skotte, Niels H., Savill, Jane, Nguyen, Yen T. N., Ladha, Safia, Cao, Li-Ping, Dullaghan, Edie, Hayden, Michael R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226564/
https://www.ncbi.nlm.nih.gov/pubmed/22140457
http://dx.doi.org/10.1371/journal.pone.0027680
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author Ehrnhoefer, Dagmar E.
Skotte, Niels H.
Savill, Jane
Nguyen, Yen T. N.
Ladha, Safia
Cao, Li-Ping
Dullaghan, Edie
Hayden, Michael R.
author_facet Ehrnhoefer, Dagmar E.
Skotte, Niels H.
Savill, Jane
Nguyen, Yen T. N.
Ladha, Safia
Cao, Li-Ping
Dullaghan, Edie
Hayden, Michael R.
author_sort Ehrnhoefer, Dagmar E.
collection PubMed
description Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders.
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spelling pubmed-32265642011-12-02 A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture Ehrnhoefer, Dagmar E. Skotte, Niels H. Savill, Jane Nguyen, Yen T. N. Ladha, Safia Cao, Li-Ping Dullaghan, Edie Hayden, Michael R. PLoS One Research Article Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders. Public Library of Science 2011-11-29 /pmc/articles/PMC3226564/ /pubmed/22140457 http://dx.doi.org/10.1371/journal.pone.0027680 Text en Ehrnhoefer et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ehrnhoefer, Dagmar E.
Skotte, Niels H.
Savill, Jane
Nguyen, Yen T. N.
Ladha, Safia
Cao, Li-Ping
Dullaghan, Edie
Hayden, Michael R.
A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
title A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
title_full A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
title_fullStr A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
title_full_unstemmed A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
title_short A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
title_sort quantitative method for the specific assessment of caspase-6 activity in cell culture
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226564/
https://www.ncbi.nlm.nih.gov/pubmed/22140457
http://dx.doi.org/10.1371/journal.pone.0027680
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