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

Andrei Zhuravlev1, Daria Ezeriņa2, Julia Ivanova1

  • 1Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii Pr. 4, St. Petersburg, 194064, Russia.

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Summary

This study repurposes the HyPer1 probe to measure cellular reduction. HyPer1 reveals distinct reduction pathways in different cell compartments, aiding oxidative stress research.

Keywords:
Disulfide bond reductionGenetically encoded biosensorsGlutathioneHyPerHydrogen peroxideKineticsRate constantsThioredoxinН(2)О(2)

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Area of Science:

  • Cellular metabolism
  • Redox biology
  • Biochemical assays

Background:

  • Cellular reduction is crucial for metabolic and regulatory processes.
  • Limited tools exist to investigate cellular reduction due to focus on oxidation.
  • HyPer1, a hydrogen peroxide (H2O2) probe, can be repurposed.

Purpose of the Study:

  • To adapt HyPer1 as a tool for measuring cellular reductive power.
  • To investigate the reduction kinetics of HyPer1 in different cellular compartments.
  • To quantify compartmentalized reduction activity following oxidative stress.

Main Methods:

  • Focused on the reduction half-reaction of HyPer1.
  • Determined HyPer1 reduction mechanisms in cytosol, nucleus, and mitochondria.
  • Utilized human leukemia K-562 cells to quantify reductive activity.
  • Assessed HyPer1 response in various human cell lines.

Main Results:

  • HyPer1 is primarily reduced by thioredoxin/thioredoxin reductase (Trx/TrxR) in cytosol and nucleus (rate constant 5.8 × 10^2 M^-1s^-1).
  • Glutathione (GSH) predominantly reduces HyPer1 in mitochondria (rate constant 1.8 M^-1s^-1).
  • Mitochondrial HyPer1 recovery was twice as long as cytosolic/nuclear recovery post-oxidative stress.
  • A potent cytosolic Trx/TrxR pathway was observed, especially in cancer cell lines.

Conclusions:

  • HyPer1 serves as a reliable tool for assessing compartmentalized reduction.
  • The study elucidates distinct reduction mechanisms across cellular compartments.
  • HyPer1 application aids in understanding cellular responses to oxidative stress.