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Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H₂O₂ via cysteine-dependent redox short-circuit.

ABSTRACT: The Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme's active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is known to increase upon SOD1 misfolding, structural alterations caused by a mutation or environmental stresses. These competing side-reactions can lead to the formation of particularly toxic ROS, which have been proposed to contribute to oxidative damage in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Here, we demonstrated that metal-saturated SOD1^WT (holo-SOD1^WT) and a familial ALS (fALS) catalytically active SOD1 mutant, SOD1^G93A, are capable, under defined metabolic circumstances, to generate cytotoxic quantities of H₂O₂ through cysteine (CSH)/glutathione (GSH) redox short-circuit. Such activity may drain GSH stores, therefore discharging cellular antioxidant potential. By analyzing the distribution of thiol compounds throughout the CNS, the location of potential hot-spots of ROS production can be deduced. These hot-spots may constitute the origin of oxidative damage to neurons in ALS.

SUBMITTER: Bakavayev S

PROVIDER: S-EPMC6658568 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H<sub>2</sub>O<sub>2</sub> via cysteine-dependent redox short-circuit.

Bakavayev Shamchal S Chetrit Nimrod N Zvagelsky Tatiana T Mansour Rasha R Vyazmensky Maria M Barak Zeev Z Israelson Adrian A Engel Stanislav S

Scientific reports 20190725 1

The Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme's active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is kno ...[more]

PMID: 31346243

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Project description:BackgroundCopper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity in macrophages exposed to CuO NPs versus copper chloride (CuCl2).MethodsThe mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay.ResultsWe observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death.ConclusionsCuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.

Dataset Information

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H₂O₂ via cysteine-dependent redox short-circuit.

Publications

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H<sub>2</sub>O<sub>2</sub> via cysteine-dependent redox short-circuit.

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Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H2O2 via cysteine-dependent redox short-circuit.

Publications

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H<sub>2</sub>O<sub>2</sub> via cysteine-dependent redox short-circuit.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H₂O₂ via cysteine-dependent redox short-circuit.