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Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells

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Abstract

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN−, a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]4 cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]4 cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]4 cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.

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Abbreviations

c-aconitase:

Cytosolic aconitase

DFO:

Desferrioxamine

EMSA:

Electrophoretic mobility shift assay

HCAEC:

Primary human coronary artery endothelial cells

HOCl:

The physiological mixture of hypochlorous acid and its anion

HOSCN:

The physiological mixture of hypothiocyanous acid and its anion

IREs:

Iron responsive elements

IRP-1:

Iron response protein-1

m-aconitase:

Mitochondrial aconitase

MPO:

Myeloperoxidase

TCA:

Trichloroacetic acid

TfR:

Transferrin receptor

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Acknowledgments

We thank Mr. Pat Pisansarakit for tissue culture support, and Drs. David Pattison, Naomi Cook, Benjamin Rayner and Frederick Luk for help and advice on experimental methods. We thank the Australian Research Council (through the Centres of Excellence Scheme, CE0561607, and Discovery Programs DP0988311), and the Novo Nordisk Foundation (Laureate Research Grant NNF13OC0004294 to MJD) for financial support.

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Authors and Affiliations

  1. The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW, 2042, Australia

    Jihan Talib & Michael J. Davies

  2. Faculty of Medicine, University of Sydney, Sydney, NSW, 2006, Australia

    Jihan Talib & Michael J. Davies

  3. Department of Biomedical Sciences, Panum Institute, University of Copenhagen, 2200, Copenhagen, Denmark

    Michael J. Davies

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  1. Jihan Talib
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Correspondence to Michael J. Davies.

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Talib, J., Davies, M.J. Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells. J Biol Inorg Chem 21, 305–317 (2016). https://doi.org/10.1007/s00775-016-1340-4

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  • DOI: https://doi.org/10.1007/s00775-016-1340-4

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