Fig 1: CuO NPs trigger unfolding of SOD1. a SOD1 is a dimeric protein comprised of an eight-stranded β-barrel with one Cu (orange) and Zn (cyan) ion bound in each monomer. Reproduced from: Trist BG, Hilton JB, Hare DJ, Crouch PJ, Double KL. Superoxide dismutase 1 in health and disease: how a frontline antioxidant becomes neurotoxic. Angew Chem Int Ed Engl. 2021;60(17):9215–46 under a Creative Commons license (CC BY 4.0). b–e CD spectroscopy measurements were performed to assess conformational changes in recombinant human SOD1. b CuO NPs decreased the α-helix and β-sheet conformation of the SOD1 protein. c CuO NPs dissolved in ALF (pH 4.5) shifted the α-helix conformation of SOD1 protein from 208 to 212 nm. d CuCl2 caused only a minor effect on the α-helical content, while e ZnCl2 showed no effect. ZnO NPs could not be studied due to assay interference
Fig 2: SOD1 expression and role of the SOD1 chaperone, MIF. a, b Western blot analysis of cells exposed for 12 h to CuO NPs at the indicated concentrations in the presence or absence of the MIF inhibitor, 4-IPP (50 µM). Membranes were probed for SOD1 and MIF, and reprobed using antibodies against ß-actin as a loading control. Cells were also exposed to the proteasome inhibitor, bortezomib (5 nM). (a) shows the results obtained under non-reducing conditions and (b) shows the results under reducing conditions (10 mM DTT added to the lysis buffer prior to the electrophoresis). Note that at high concentrations of CuO NPs (50 µg/mL) the loading control was also affected (under non-reducing conditions) and therefore the loss of the SOD1 monomer at this concentration cannot be evaluated. In cells exposed to CuO NPs under non-reducing conditions, high molecular weight SOD1 oligomers appeared which disappeared when the samples were run under reducing conditions (data not shown). Similar results were obtained in several experiments performed at 12 h or 24 h. c–f Pharmacological inhibition of the SOD1 chaperone, MIF aggravates CuO NP-induced cell death. RAW264.7 macrophages were exposed for 6 h to medium alone (c), 4-IPP (50 µM) (d), or 4-IPP plus CuO NPs (10 µg/mL) (e) and confocal microscopy was performed to assess the localization of SOD1 (green). Mitochondria and nuclei were identified using MitoTracker™ Red and DAPI (blue), respectively. f Cells were exposed for 24 h to CuO NPs at the indicated concentrations in the presence or absence of the MIF inhibitor, 4-IPP (50 µM) and cell viability (metabolic capacity) was determined using the Alamar blue assay. ****p < 0.0001; ####p < 0.0001
Fig 3: SOD1 redistribution in CuO NP-exposed cells. SOD1 localization in RAW264.7 cells was determined by confocal microscopy. SOD1 was visualized using a specific antibody and an Alexa Fluor® 488-conjugated secondary antibody (green). Mitochondria were detected using MitoTracker™ Red and cell nuclei were counterstained using DAPI (blue). a Control; b CuO NPs (10 µg/mL); c CuO NPs (25 µg/mL); d CuCl2 (50 µg/mL). The cells were exposed for 6 h
Fig 4: Current model of CuO NP-triggered cell death. The figure depicts cellular uptake of CuO NPs by macrophages leading to their deposition in lysosomes. This is followed by the dissolution of the NPs at acidic pH with subsequent effects of copper ions on the cells leading to protein misfolding and mitochondrial dysfunction with ensuing oxidative stress. Furthermore, we have demonstrated proteasome inhibition in cells exposed to CuO NPs. This could potentially be due to massive protein misfolding leading to proteasomal insufficiency and/or to a direct inhibitory effect of Cu on the proteasome. MIF is an SOD1 chaperone. Consult text for details
Supplier Page from Abcam for Recombinant human Superoxide Dismutase 1 protein (Active)