Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton

Our plain-English summary of Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton, by K.J. Nelson, T. Messier, S. Milczarek, et al (Antioxidants, 2021).

The study

• Authors: K.J. Nelson, T. Messier, S. Milczarek, A. Saaman, S. Beuschel, U. Gandhi, N. Heintz, T.L. Smalley, W.T. Lowther, B. Cunniff
• Journal: Antioxidants
• Published: 2021;10(2)
• Cited by: 12 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1-5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.

In plain English

This laboratory study examined why the antibiotic thiostrepton (TS) reacts specifically with mitochondrial peroxiredoxin 3 (PRX3). Working with primary and immortalized normal mesothelial cells and patient-derived malignant mesothelioma (MM) cell lines, the authors found MM lines more sensitive to TS (EC50 about 0.9-2.4 micromolar) than normal mesothelial cells (about 2.8-4.7 micromolar), and reported a linear relationship between TS EC50 and PRX3 protein expression (slope significantly non-zero, p = 0.025). MitoSOX Red staining, which primary mesothelial cells were not amenable to, was higher in most MM lines than in LP9 normal mesothelial cells and also related linearly to TS EC50 (p = 0.028). In MM cells TS produced a dose-dependent non-reducible PRX3 dimer, with non-significant changes to PRX1 and PRX2 and no observable effect on PRX4. With recombinant PRXs 1-5 at pH 8.0, PRXs 1-3 formed 12-23% crosslinked dimer, PRX4 showed no significant crosslink and PRX5 none. Engineered dimers reacted more at pH 8 (20-50% adduct) than pH 7 (0-7%). Mass spectrometry indicated TS reacted with the reduced thiol, not the sulfenic acid. Gentian violet potentiated PRX3 adduction; disrupting membrane potential did not affect TS activity.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study combining cell-culture experiments in mesothelioma cell lines with biochemical and biophysical experiments on recombinant human peroxiredoxins
• Who was studied: A panel of primary mesothelial, immortalized non-tumorigenic mesothelial and patient-derived malignant mesothelioma cell lines (pleural and peritoneal, sarcomatoid and biphasic subtypes), plus recombinant human PRX1-PRX5 proteins and engineered dimeric variants
• Exposure: Treatment of cells and purified proteins with thiostrepton, alone or with gentian violet or the uncoupler FCCP; in vitro turnover with hydrogen peroxide at pH values from cytosolic to mitochondrial
• Main results:
  • Primary and immortalized non-tumorigenic mesothelial cells had TS EC50 values of about 2.8-4.7 micromolar
  • Malignant mesothelioma cell lines had TS EC50 values of about 0.9-2.4 micromolar
  • At their respective EC99 concentrations, MM cell lines were on average about 10 times more sensitive to TS than normal mesothelial cells
  • TS sensitivity (EC50) showed a linear relationship with PRX3 protein expression across the cell lines; slope significantly non-zero (p = 0.025)
  • MitoSOX Red fluorescence was increased in most MM lines compared with LP9 normal mesothelial cells, and TS EC50 related linearly to MitoSOX Red signal; slope significantly non-zero (p = 0.028). Primary mesothelial cells could not be assessed with this dye
  • Mitochondrial spare reserve capacity was lower in MM cells than normal mesothelial cells but did not significantly correlate with TS sensitivity
  • In MM cells, TS produced a dose-dependent non-reducible PRX3 dimer, with non-significant changes to PRX1 and PRX2 and no observable effect on PRX4
  • With recombinant proteins cycled at pH 8.0, PRXs 1-3 formed 12-23% crosslinked dimer; no significant crosslink for PRX4 and no adduct detected for PRX5
  • Engineered dimers of PRX1, PRX2 and PRX3 were more reactive with TS at higher pH (20-50% adduct at pH 8 versus 0-7% at pH 7)
  • Wild-type PRXs 1-3 gave 5-15% PRX-TS-PRX adduct at mitochondrial pH values versus 2-5% at cytosolic pH values
  • Mass spectrometry showed a mass increase consistent with one TS molecule (1660 Da, theoretical 1664 Da) only for the reduced PRX3 variant, not the sulfenic acid form
  • By SEC-MALS, oxidized PRX3 was fully dimeric at pH 7.5 and 8.0, while oxidized PRX1 remained predominantly decameric and PRX2 retained significant decamer even when fully oxidized
  • PRX3 was fully dimeric after oxidation of 6 of its 12 active sites, with dimer appearing after 1/6 equivalents of hydrogen peroxide
  • Gentian violet (1 micromolar, 24 h) increased mitochondrial PRX3 disulfide-bonded dimers with no observable effect on PRX1, PRX2 or PRX4, and increased PRX3 crosslinking by TS dose-dependently
  • FCCP-mediated loss of mitochondrial membrane potential did not change TS crosslinking of PRX3 at 4 or 24 h or TS cytotoxicity, whereas it blunted gentian violet effects
• The authors' conclusion: The authors concluded that the specificity of TS for PRX3 is driven by its mitochondrial location with higher pH plus structural features that resist hyperoxidation and favour collapse of the dodecamer to dimer, supporting further development of this prooxidant strategy
• Limitations the authors state:
  • PRX3 could not be tested at pH 7.0 because of instability and precipitation at that pH
  • Oxidation state is only one determinant; the dimer/(do)decamer equilibrium is also influenced by protein concentration, salt, pH and affinity or reporter tags
  • The mechanism by which TS enters the mitochondria is unknown
  • Experiments used a constant 100 micromolar PRX concentration, described as somewhat low for mitochondrial PRX3 estimates
  • Primary mesothelial cells could not be assessed with MitoSOX Red because they lifted from the plate

Topics

Medical terms in this study, explained in our Lexicon: Carcinogenesis, Reactive Oxygen Species (ROS), Mesothelioma.

Related studies

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• Mechanism-based peroxiredoxin 3 inhibitors exploit a covalent warhead for cancer therapy — K.J. Nelson, T.L. Smalley, et al. (2025)
• Preclinical characterization and phase 1 clinical testing of targeting mitochondrial peroxiredoxin 3 in cancer — V. Gibson, J. Dzialo, et al. (2026)
• Cellular and molecular basis of the asbestos-related diseases — A. Cosentino, M.L. Warnock (1992)
• Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma — E. Lou, S. Fujisawa, et al. (2012)
• Peroxiredoxin 3 levels regulate a mitochondrial redox setpoint in malignant mesothelioma cells — B. Cunniff, A.N. Wozniak, et al. (2014)

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