Dataset Information

Protein dynamics of [Cu-Zn] superoxide dismutase (SOD1): How protein motions at the global and local levels impact the reactivity of SOD1.

ABSTRACT: This work explores the pivotal role that protein mobility plays in facilitating the catalytic activity of Copper-Zinc superoxide dismutase (SOD1). Through both localized active site distortions and correlated domain movement, these motions enable the enzyme to adopt the conformations necessary to achieve both substrate delivery and efficient catalytic transformation. Structural and computational studies of a biomimetic model complex are used to probe the localized interactions between substrate and secondary sphere residues that play a role in guiding substrate to the active site, as well as facilitating the conformational changes necessary for substrate turnover. Normal mode analysis (NMA) of SOD1 demonstrates how collective domain motion influences key residues of the electrostatic loop (ESL), guiding substrate to the active site and facilitating the delivery of the conserved water network necessary for proton transfer.

SUBMITTER: Healy EF

PROVIDER: S-EPMC7586375 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Protein dynamics of [Cu-Zn] superoxide dismutase (SOD1): How protein motions at the global and local levels impact the reactivity of SOD1.

Healy Eamonn F EF Flores Rafael R Lynch Vincent M VM Toledo Santiago S

Journal of inorganic biochemistry 20200624

This work explores the pivotal role that protein mobility plays in facilitating the catalytic activity of Copper-Zinc superoxide dismutase (SOD1). Through both localized active site distortions and correlated domain movement, these motions enable the enzyme to adopt the conformations necessary to achieve both substrate delivery and efficient catalytic transformation. Structural and computational studies of a biomimetic model complex are used to probe the localized interactions between substrate ...[more]

PMID: 32619897

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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

Protein dynamics of [Cu-Zn] superoxide dismutase (SOD1): How protein motions at the global and local levels impact the reactivity of SOD1.

Publications

Protein dynamics of [Cu-Zn] superoxide dismutase (SOD1): How protein motions at the global and local levels impact the reactivity of SOD1.

Similar Datasets

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