HyPer as a tool to determine the reductive activity in cellular compartments

Andrei Zhuravlev, Daria Ezeriņa, Julia Ivanova, Nikita Guriev, Natalia Pugovkina, Alla Shatrova, Nikolay Aksenov, Joris Messens, Olga Lyublinskaya

Research output: Contribution to journalArticlepeer-review


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 H 2O 2 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 × 10 2 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.

Original languageEnglish
Article number103058
Number of pages33
JournalRedox Biology
Publication statusPublished - Apr 2024

Bibliographical note

Funding Information:
The authors are thankful to Prof. Fernando Antunes (University of Lisbon, Portugal) for stimulating ideas and discussions. O.L. was supported by the Russian Science Foundation (Grant No. 21-74-20178 ). J.M. and D.E. are supported by a VIB grant and are grateful to Elias Arnér and Qing Cheng for providing the hTrx1 and hTrx2 expression constructs and to Carsten Berndt for the provision of the hGrx1 and hGrx2 constructs.

Publisher Copyright:
© 2024 The Authors


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