Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells
Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase proc...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-05-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/11/6/169 |
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| author | Mei C. Q. Houser Yuliia Turchyna Florian Perrin Lori Chibnik Oksana Berezovska Masato Maesako |
| author_facet | Mei C. Q. Houser Yuliia Turchyna Florian Perrin Lori Chibnik Oksana Berezovska Masato Maesako |
| author_sort | Mei C. Q. Houser |
| collection | DOAJ |
| description | Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells. |
| first_indexed | 2024-03-10T11:02:41Z |
| format | Article |
| id | doaj.art-a33e9e7e9c9540cf8abd6d6be3653847 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-10T11:02:41Z |
| publishDate | 2021-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-a33e9e7e9c9540cf8abd6d6be36538472023-11-21T21:23:17ZengMDPI AGBiosensors2079-63742021-05-0111616910.3390/bios11060169Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live CellsMei C. Q. Houser0Yuliia Turchyna1Florian Perrin2Lori Chibnik3Oksana Berezovska4Masato Maesako5Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USAPresenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells.https://www.mdpi.com/2079-6374/11/6/169PS/γ-secretaseFRETmultiplexingAPP C99Notch1 N100 |
| spellingShingle | Mei C. Q. Houser Yuliia Turchyna Florian Perrin Lori Chibnik Oksana Berezovska Masato Maesako Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells Biosensors PS/γ-secretase FRET multiplexing APP C99 Notch1 N100 |
| title | Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells |
| title_full | Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells |
| title_fullStr | Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells |
| title_full_unstemmed | Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells |
| title_short | Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells |
| title_sort | limited substrate specificity of ps γ secretase is supported by novel multiplexed fret analysis in live cells |
| topic | PS/γ-secretase FRET multiplexing APP C99 Notch1 N100 |
| url | https://www.mdpi.com/2079-6374/11/6/169 |
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