Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells.
dimer-decamer equilibrium
fluorescence polarization
homo-FRET
peroxiredoxins
protein oligomerization
Journal
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Titre abrégé: Proc Natl Acad Sci U S A
Pays: United States
ID NLM: 7505876
Informations de publication
Date de publication:
14 07 2020
14 07 2020
Historique:
pubmed:
1
7
2020
medline:
24
9
2020
entrez:
1
7
2020
Statut:
ppublish
Résumé
Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.
Identifiants
pubmed: 32601209
pii: 1915275117
doi: 10.1073/pnas.1915275117
pmc: PMC7368258
doi:
Substances chimiques
Homeodomain Proteins
0
Luminescent Proteins
0
PRRX2 protein, human
0
Recombinant Fusion Proteins
0
Hydrogen Peroxide
BBX060AN9V
2-cys peroxiredoxin, human
EC 1.11.1.15
Peroxiredoxins
EC 1.11.1.15
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
16313-16323Commentaires et corrections
Type : CommentIn
Informations de copyright
Copyright © 2020 the Author(s). Published by PNAS.
Déclaration de conflit d'intérêts
The authors declare no competing interest.
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