A tangible method to assess native ferroptosis suppressor activity
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved..
Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:4 |
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| Enthalten in: |
Cell reports methods - 4(2024), 3 vom: 25. März, Seite 100710 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Nakamura, Toshitaka [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 28.03.2024 Date Revised 04.04.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.crmeth.2024.100710 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 245 | 1 | 2 | |a A tangible method to assess native ferroptosis suppressor activity |
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| 520 | |a Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms | ||
| 650 | 4 | |a Journal Article | |
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| 650 | 4 | |a LC-MS/MS | |
| 650 | 4 | |a RSL3 | |
| 650 | 4 | |a affinity purification | |
| 650 | 4 | |a biochemistry | |
| 650 | 4 | |a cell death | |
| 650 | 4 | |a drug discovery | |
| 650 | 4 | |a enzyme assay | |
| 650 | 4 | |a lipid peroxidation | |
| 650 | 4 | |a pull-down assay | |
| 650 | 4 | |a selenocysteine | |
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| 700 | 1 | |a Ito, Junya |e verfasserin |4 aut | |
| 700 | 1 | |a Mourão, André Santos Dias |e verfasserin |4 aut | |
| 700 | 1 | |a Wahida, Adam |e verfasserin |4 aut | |
| 700 | 1 | |a Nakagawa, Kiyotaka |e verfasserin |4 aut | |
| 700 | 1 | |a Mishima, Eikan |e verfasserin |4 aut | |
| 700 | 1 | |a Conrad, Marcus |e verfasserin |4 aut | |
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