SOD1 oxidation and formation of soluble aggregates in yeast: relevance to sporadic ALS development

Abstract

Misfolding and aggregation of copper-zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS.

Keywords: Oxidative PTMs; Soluble aggregates; Sporadic ALS; Wild-type Sod1; Yeast.

Graphical abstract

Fig. 1

Active-site residues in Sod1. The catalytic Cu cofactor is coordinated to His46, His48, His120 and bridging His63. The structural Zn cofactor is coordinated to His63, His71 and His80 as well as aspartic acid Asp83 (not shown). The intrasubunit disulfide bridge between Cys57 and Cys146 is important in stabilizing the Sod1 homodimer [7,8,20]. The distances from Ne of His120 and His71, and the Cys57–Cys146 disulfide bridge to the catalytic Cu (2.1, 6.9 and 9.5 Å, respectively) were measured in the 1.15-Å resolution crystal structure of human Sod1 (PDB 2V0A).

Fig. 2

SEC reveals the presence of HMW and LMW Sod1 populations in the soluble protein extract from 7-day stationary-phase yeast cells. (A) Size exclusion chromatogram of the extract. A 100-µL aliquot of extract containing 0.4 mg of protein was diluted to 1 mL with 20 mM KPi (pH 7.0) and loaded on a Superose 12 HR 10/30 column (1.0 cm × 30 cm) equilibrated with 20 mM KPi/300 mM NaCl (pH 7.0) and connected to the ÄKTApurifier. Proteins were eluted with the equilibration buffer at a flow rate of 0.3 mL/min and detected at 280 nm. The arrows indicate the fractions that were tested for immunoreactivity and SOD activity. (B) Upper panel: in-gel Sod1 activity following native PAGE of the extract and the SEC fractions each containing 1 µg of total protein. Bands were stained with riboflavin as a superoxide generator and NBT, which is reduced to formazan in the presence of superoxide [32]. (B) Lower panel: Sod1 protein was detected by immunodot blot in the extract, two fractions of the SEC column void volume P1 (7 mL) and P1' (7.5 mL), and in P3 (18 mL). Samples containing 10 µg of protein were dotted onto PVDF membranes and probed with rabbit anti-human Sod1 antibody. See "Materials and methods" section for additional information. Sod1 that was eluted in P1, P1' and in P3 is referred to in the text as HMW Sod1 and LMW Sod1, respectively. P1 and P1' were combined and treated as a single P1 fraction for LC–MS analysis. Proteins were extracted from three independent cultures and representative results are presented here.

Fig. 3

Decomplexation of Sod1-containing SEC fractions by SDS-PAGE. Aliquots (˜25 µL) of the P1 and P3 SEC fractions from Fig. 2A containing 20 µg of protein were analyzed by reducing SDS-PAGE. Bands were excised from lanes P1 to P3 of the gel and subjected to in-gel tryptic digestion. Digests from the R15 and R25 regions (denoted by the red boxes) gave the highest score and Sod1 sequence coverage so Tables 2 and 3 summarize the LC–MS/MS results for these regions. Extracts from three independent cultures were analyzed and the gel shown is representative of the three. Blue lines indicate the centers of the MW markers, which are poorly visible in the scanned gel (lane MW).

Fig. 4

HMW Sod1 is oxidized at Cys146, His71 and His120. MS/MS spectrum of the (A) (M + 2 H)²⁺ ion at m/z 823.41 of the C-terminal peptide containing Cys146-SO₃H; (B) (M + 3 H)³⁺ ion at m/z 738.70 of the oxo-His120 peptide; and (C) (M + 3 H)³⁺ ion at m/z 366.84 of the oxo-His71 peptide. The peptide (precursor) ions (green) (see Table S1 and sequence above spectra) were fragmented by CID (30 V) to give b (red) and y sequence ions (blue). The smallest visible b or y ion bearing the modified residue (see Tables S2–S4) is circled in each spectrum. The peptide ion match (number of y+b ions identified in the MS/MS spectrum divided by the theoretical number of y+b ions generated by CID) is (A) 18/32, (B) 22/80, and (C) 13/36. The XCorr values are (A) 2.5, (B) 2.0 and (C) 2.0. The MS operating parameters are given in the "Materials and methods" section.

Fig. 5

HMW Sod1-GFP accumulates as yeast age but declines in very old cells. Soluble protein extracts from cells chromosomally expressing Sod1-GFP were fractionated by SEC as described in the legend of Fig. 2 except that the Superose column was coupled to an Agilent 1100 HPLC with a fluorescence detector, and GFP fluorescence was monitored with ex/em 470/520 nm. (A) Size-exclusion chromatogram of extracts from 3- (black line) and 7-day (green area) cells. LMW Sod1-GFP is present in the intense peaks centered at ˜11 mL and the inset shows a 100-fold expansion of the HMW Sod1-GFP fractions at 6.5 mL. (B) Variation with cell age of HMW Sod1-GFP as a percentage of the total peak area.