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Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase
Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-pep...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663845/ https://www.ncbi.nlm.nih.gov/pubmed/23717518 http://dx.doi.org/10.1371/journal.pone.0063962 |
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author | Schedin-Weiss, Sophia Inoue, Mitsuhiro Teranishi, Yasuhiro Yamamoto, Natsuko Goto Karlström, Helena Winblad, Bengt Tjernberg, Lars O. |
author_facet | Schedin-Weiss, Sophia Inoue, Mitsuhiro Teranishi, Yasuhiro Yamamoto, Natsuko Goto Karlström, Helena Winblad, Bengt Tjernberg, Lars O. |
author_sort | Schedin-Weiss, Sophia |
collection | PubMed |
description | Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies. |
format | Online Article Text |
id | pubmed-3663845 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-36638452013-05-28 Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase Schedin-Weiss, Sophia Inoue, Mitsuhiro Teranishi, Yasuhiro Yamamoto, Natsuko Goto Karlström, Helena Winblad, Bengt Tjernberg, Lars O. PLoS One Research Article Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies. Public Library of Science 2013-05-24 /pmc/articles/PMC3663845/ /pubmed/23717518 http://dx.doi.org/10.1371/journal.pone.0063962 Text en © 2013 Schedin-Weiss 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 Schedin-Weiss, Sophia Inoue, Mitsuhiro Teranishi, Yasuhiro Yamamoto, Natsuko Goto Karlström, Helena Winblad, Bengt Tjernberg, Lars O. Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase |
title | Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase |
title_full | Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase |
title_fullStr | Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase |
title_full_unstemmed | Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase |
title_short | Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase |
title_sort | visualizing active enzyme complexes using a photoreactive inhibitor for proximity ligation – application on γ-secretase |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663845/ https://www.ncbi.nlm.nih.gov/pubmed/23717518 http://dx.doi.org/10.1371/journal.pone.0063962 |
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