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Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1-Cn protein interactions occurring in vitro and in vivo.

ABSTRACT: Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2*-). The effects of O2*- on Cn are still controversial. We found that O2*-, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1-rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2*- dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase K(m) without a change in V(max). A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1-Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.

SUBMITTER: Agbas A

PROVIDER: S-EPMC1925239 | biostudies-other | 2007 Jul

REPOSITORIES: biostudies-other

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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.

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Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1-Cn protein interactions occurring in vitro and in vivo.

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