Cargando…
Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin
Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Her...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711817/ https://www.ncbi.nlm.nih.gov/pubmed/33255272 http://dx.doi.org/10.3390/mps3040081 |
_version_ | 1783618229847982080 |
---|---|
author | Suire, Caitlin N. Brizuela, Monica K. Leissring, Malcolm A. |
author_facet | Suire, Caitlin N. Brizuela, Monica K. Leissring, Malcolm A. |
author_sort | Suire, Caitlin N. |
collection | PubMed |
description | Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications. |
format | Online Article Text |
id | pubmed-7711817 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-77118172020-12-04 Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin Suire, Caitlin N. Brizuela, Monica K. Leissring, Malcolm A. Methods Protoc Article Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications. MDPI 2020-11-24 /pmc/articles/PMC7711817/ /pubmed/33255272 http://dx.doi.org/10.3390/mps3040081 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Suire, Caitlin N. Brizuela, Monica K. Leissring, Malcolm A. Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin |
title | Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin |
title_full | Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin |
title_fullStr | Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin |
title_full_unstemmed | Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin |
title_short | Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin |
title_sort | quantitative, high-throughput assays for proteolytic degradation of amylin |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711817/ https://www.ncbi.nlm.nih.gov/pubmed/33255272 http://dx.doi.org/10.3390/mps3040081 |
work_keys_str_mv | AT suirecaitlinn quantitativehighthroughputassaysforproteolyticdegradationofamylin AT brizuelamonicak quantitativehighthroughputassaysforproteolyticdegradationofamylin AT leissringmalcolma quantitativehighthroughputassaysforproteolyticdegradationofamylin |