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The Reducible Disulfide Proteome of Synaptosomes Supports a Role for Reversible Oxidations of Protein Thiols in the Maintenance of Neuronal Redox Homeostasis

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Abstract

The mechanisms by which neurons maintain redox homeostasis, disruption of which is linked to disease, are not well known. Hydrogen peroxide, a major cellular oxidant and neuromodulator, can promote reversible oxidations of protein thiols but the scope, targets, and significance of such oxidations occurring in neurons, especially in vivo, are uncertain. Using redox phenylarsine oxide (PAO)-affinity chromatography, which exploits the high-affinity of trivalent arsenicals for protein dithiols, this study investigated the occurrence of reducible and, therefore, potentially regulatory, protein disulfide bonds in Triton X-100-soluble protein fractions from isolated nerve-endings (synaptosomes) prepared from rat brains. Postmortem oxidations of protein thiols were limited by rapidly freezing the brains following euthanasia and, later, homogenizing them in the presence of the N-ethylmaleimide to trap reduced thiols. The reducible disulfide proteome comprised 5.4% of the total synaptosomal protein applied to the immobilized PAO columns and was overrepresented by pathways underlying ATP synaptic supply and demand including synaptic vesicle trafficking. The alpha subunits of plasma membrane Na+, K+-ATPase and the mitochondrial ATP synthase were particularly abundant proteins of the disulfide proteome and were enriched in this fraction by 3.5- and 6.7-fold, respectively. An adaptation of the commonly used “biotin-switch” method provided additional support for selective oxidation of thiols on the alpha subunit of the ATP synthase. We propose that reversible oxidations of protein thiols may underlie a coordinated metabolic response to hydrogen peroxide, serving to both control redox signaling and protect neurons from oxidant stress.

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Abbreviations

Biotin-HPDP:

N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide

CaN:

Calcineurin

DTT:

Dithiothreitol

FT:

Flow-through

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GSH:

Glutathione

KHH:

Krebs–Henseleit–HEPES

LW:

Last wash

LC–MS/MS:

Liquid chromatography-tandem mass spectrometry

NEM:

N-ethylmaleimide

NNT:

Nicotinamide nucleotide transhydrogenase

PAO:

Phenylarsine oxide

PPP:

Pentose phosphate pathway

ROS:

Reactive oxygen species

TCEP:

Tris(2-carboxyethyl)phosphine

Trx:

Thioredoxin

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Acknowledgements

The authors thank the University of Scranton for the financial support for this work. They also thank Ms. Courtney Higgins for technical support and Mr. Richard Trygar for administrative support and of this project.

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This work was not supported by external funding.

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Authors and Affiliations

  1. Department of Chemistry, University of Scranton, Scranton, PA, 18510, USA

    Timothy D. Foley, Giancarlo Montovano & Monserrath Camacho Ayala

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  1. Timothy D. Foley
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Contributions

TDF designed the study, supervised the performance of the work and the collection and analyses of data, and wrote most of the manuscript. GM and MCA performed all of the laboratory work, including refinement of methods, and contributed to data analyses and manuscript preparation. All authors approved the final paper.

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Correspondence to Timothy D. Foley.

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The study was approved by the Institutional Animal Care and Use Committee of the University of Scranton (2019, Protocol #7-19).

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Foley, T.D., Montovano, G. & Camacho Ayala, M. The Reducible Disulfide Proteome of Synaptosomes Supports a Role for Reversible Oxidations of Protein Thiols in the Maintenance of Neuronal Redox Homeostasis. Neurochem Res 45, 1825–1838 (2020). https://doi.org/10.1007/s11064-020-03046-7

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