Figure 1.
ApoSOD1 variants displaying reduced cytotoxicity migrate slower in non-reducing SDS-PAGE.
ApoSOD1 in dimeric or monomeric form were added to the cell media of cultured SH-SY5Y cells and incubated for 72 h. Proteins were added in duplicate wells and cell viability was measured with the MTT assay. Cell viability is presented as the mean percentage viability of the buffer control ± range. (A) Dimeric wild-type apoSOD1 induced very little toxicity, whereas cysteine alkylation with iodoacetamide (IAM) or substitution of C111 for alanine restored toxicity almost, but not completely, to the level of apoSOD1 C6/111A. (B) Only monomeric apoSOD1 with C6 or both C6 and C111 substituted for alanine were toxic, whereas wild-type, wild-type (IAM) and C111A were essentially non-toxic. (C) Non-reducing and reducing SDS-PAGE of apoSOD1 samples before adding them to the cell culture. All proteins mainly migrate as native SOD1 (compare with C6/111A). (D) Non-reducing and reducing SDS-PAGE of apoSOD1 samples collected from the cell culture after 72 h of incubation. ApoSOD1 variants displaying reduced toxicity all migrate with a slower rate under non-reducing conditions compared to the toxic proteins C6/111A and C6A. Under reducing conditions, all proteins migrate with the same rate. (E) The band intensity of the native band was clearly correlated to cell viability. The cellular response for each apoSOD1 variant was determined by the area under the viability curve in A and B.
Figure 2.
Monomeric SOD1 variants with slow migration rate in non-reducing SDS-PAGE aggregate into ThT positive aggregates.
ApoSOD1 (90 μM) was aggregated with 10 μM ThT in PBS without agitation for 83h (4950 min). Data points were plotted with 50 min interval. Each data point represents the mean of three separate experiments run in duplicate. Individual aggregation curves are presented in Figure S5. (A) None of the dimeric proteins aggregate under these conditions. (B) Monomeric C111A display a high ThT signal. Also wild-type and wild-type (IAM) aggregate into ThT positive aggregates, with a somewhat higher signal for wild-type (IAM).
Figure 3.
SOD1 oligomers are stabilized by intermolecular disulfide formation.
(A) Non-reducing and (B) reducing SDS-PAGE of aggregated apoSOD1 collected after 83 h of incubation in PBS without agitation. Monomeric apoSOD1 wild-type, C111A and wild-type (IAM) all form both low molecular weight and high molecular weight species stabilized by inter-molecular disulfide cross-linking as demonstrated by their disappearance in the reducing gel. All dimeric apoSOD1 variants mainly migrate as native monomer on the non-reducing gel with only very weak staining for higher molecular weight species.
Figure 4.
Electron micrographs of aggregated monomeric apoSOD1.
Aggregated monomeric apoSOD1 (on a F50E/G51E background) was applied to carbon-coated copper grids and stained with uranyl acetate. (A) wild-type (B) C6A (C) C111A (D) wild-type (IAM) (E) C6/111A (F) negative control (PBS). Monomeric apoSOD1 C111A and wild-type (IAM) contained a large amount of distinct curvilinear oligomers, whereas the aggregates formed from apoSOD1 wild-type were more “fuzzy” and clustered together. Small oligomers in less abundance were observed for apoSOD1 C6A and C6/111A.
Figure 5.
Schematic representation of the effect of disulfide scrambling on the Zn2+ chelating capacity and aggregation of apoSOD1.
ApoSOD1 chelate Zn2+ from the culture media, thereby draining the cells of this essential metal and reducing cell viability. Formation of non-native disulfide bonds between C6/C111 and C146 (alt. C57) reduce the Zn2+-chelating capacity of apoSOD1 and sets the protein up for aggregation into soluble oligomers stabilized by inter-molecular disulfides.