HyPer as a tool to determine the reductive activity in cellular compartments
A multitude of cellular metabolic and regulatory processes rely on controlled thiol reduction and oxidation mechanisms. Due to our aerobic environment, research preferentially focuses on oxidation processes, leading to limited tools tailored for investigating cellular reduction. Here, we advocate fo...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2024-04-01
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| Series: | Redox Biology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S221323172400034X |
| _version_ | 1827345414891765760 |
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| author | Andrei Zhuravlev Daria Ezeriņa Julia Ivanova Nikita Guriev Natalia Pugovkina Alla Shatrova Nikolay Aksenov Joris Messens Olga Lyublinskaya |
| author_facet | Andrei Zhuravlev Daria Ezeriņa Julia Ivanova Nikita Guriev Natalia Pugovkina Alla Shatrova Nikolay Aksenov Joris Messens Olga Lyublinskaya |
| author_sort | Andrei Zhuravlev |
| collection | DOAJ |
| description | A multitude of cellular metabolic and regulatory processes rely on controlled thiol reduction and oxidation mechanisms. Due to our aerobic environment, research preferentially focuses on oxidation processes, leading to limited tools tailored for investigating cellular reduction. Here, we advocate for repurposing HyPer1, initially designed as a fluorescent probe for H2O2 levels, as a tool to measure the reductive power in various cellular compartments. The response of HyPer1 depends on kinetics between thiol oxidation and reduction in its OxyR sensing domain. Here, we focused on the reduction half-reaction of HyPer1. We showed that HyPer1 primarily relies on Trx/TrxR-mediated reduction in the cytosol and nucleus, characterized by a second order rate constant of 5.8 × 102 M−1s−1. On the other hand, within the mitochondria, HyPer1 is predominantly reduced by glutathione (GSH). The GSH-mediated reduction rate constant is 1.8 M−1s−1. Using human leukemia K-562 cells after a brief oxidative exposure, we quantified the compartmentalized Trx/TrxR and GSH-dependent reductive activity using HyPer1. Notably, the recovery period for mitochondrial HyPer1 was twice as long compared to cytosolic and nuclear HyPer1. After exploring various human cells, we revealed a potent cytosolic Trx/TrxR pathway, particularly pronounced in cancer cell lines such as K-562 and HeLa. In conclusion, our study demonstrates that HyPer1 can be harnessed as a robust tool for assessing compartmentalized reduction activity in cells following oxidative stress. |
| first_indexed | 2024-03-07T23:06:21Z |
| format | Article |
| id | doaj.art-a800d409ee7243d7a7d7e9d66ec2caae |
| institution | Directory Open Access Journal |
| issn | 2213-2317 |
| language | English |
| last_indexed | 2024-03-07T23:06:21Z |
| publishDate | 2024-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Redox Biology |
| spelling | doaj.art-a800d409ee7243d7a7d7e9d66ec2caae2024-02-22T04:52:19ZengElsevierRedox Biology2213-23172024-04-0170103058HyPer as a tool to determine the reductive activity in cellular compartmentsAndrei Zhuravlev0Daria Ezeriņa1Julia Ivanova2Nikita Guriev3Natalia Pugovkina4Alla Shatrova5Nikolay Aksenov6Joris Messens7Olga Lyublinskaya8Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, RussiaVIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, BelgiumDepartment of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, RussiaDepartment of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, RussiaDepartment of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, RussiaDepartment of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, RussiaDepartment of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, RussiaVIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Corresponding author. VIB-VUB Center for Structural Biology, Redox Signaling Lab, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussel, Belgium.Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, Russia; Corresponding author. Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, 194064 St-Petersburg, Russia.A multitude of cellular metabolic and regulatory processes rely on controlled thiol reduction and oxidation mechanisms. Due to our aerobic environment, research preferentially focuses on oxidation processes, leading to limited tools tailored for investigating cellular reduction. Here, we advocate for repurposing HyPer1, initially designed as a fluorescent probe for H2O2 levels, as a tool to measure the reductive power in various cellular compartments. The response of HyPer1 depends on kinetics between thiol oxidation and reduction in its OxyR sensing domain. Here, we focused on the reduction half-reaction of HyPer1. We showed that HyPer1 primarily relies on Trx/TrxR-mediated reduction in the cytosol and nucleus, characterized by a second order rate constant of 5.8 × 102 M−1s−1. On the other hand, within the mitochondria, HyPer1 is predominantly reduced by glutathione (GSH). The GSH-mediated reduction rate constant is 1.8 M−1s−1. Using human leukemia K-562 cells after a brief oxidative exposure, we quantified the compartmentalized Trx/TrxR and GSH-dependent reductive activity using HyPer1. Notably, the recovery period for mitochondrial HyPer1 was twice as long compared to cytosolic and nuclear HyPer1. After exploring various human cells, we revealed a potent cytosolic Trx/TrxR pathway, particularly pronounced in cancer cell lines such as K-562 and HeLa. In conclusion, our study demonstrates that HyPer1 can be harnessed as a robust tool for assessing compartmentalized reduction activity in cells following oxidative stress.http://www.sciencedirect.com/science/article/pii/S221323172400034XDisulfide bond reductionThioredoxinGlutathioneHydrogen peroxideН2О2Genetically encoded biosensors |
| spellingShingle | Andrei Zhuravlev Daria Ezeriņa Julia Ivanova Nikita Guriev Natalia Pugovkina Alla Shatrova Nikolay Aksenov Joris Messens Olga Lyublinskaya HyPer as a tool to determine the reductive activity in cellular compartments Redox Biology Disulfide bond reduction Thioredoxin Glutathione Hydrogen peroxide Н2О2 Genetically encoded biosensors |
| title | HyPer as a tool to determine the reductive activity in cellular compartments |
| title_full | HyPer as a tool to determine the reductive activity in cellular compartments |
| title_fullStr | HyPer as a tool to determine the reductive activity in cellular compartments |
| title_full_unstemmed | HyPer as a tool to determine the reductive activity in cellular compartments |
| title_short | HyPer as a tool to determine the reductive activity in cellular compartments |
| title_sort | hyper as a tool to determine the reductive activity in cellular compartments |
| topic | Disulfide bond reduction Thioredoxin Glutathione Hydrogen peroxide Н2О2 Genetically encoded biosensors |
| url | http://www.sciencedirect.com/science/article/pii/S221323172400034X |
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