In all variants except A4V SOD1, glutathionylation significantly increases the formation of the oligomeric population eluting just prior to the native-like dimer: O1, the putative expanded dimer (Figures ?(Numbers2B2B and ?and1D).1D). Current evidence helps the pathogenic capacity of soluble misfolded SOD1, rather than the large insoluble aggregates that appear only near the onset of paralysis in ALS mouse models.3?7 However, little is known about the structural features of soluble non-native SOD1 conformers or the factors in the cellular environment that influence misfolding and aggregation. Soluble misfolded WT SOD1 has been found in the spinal cord from sporadic ALS individuals that do not carry mutations in Glutathionylation of SOD1 SOD1 was glutathionylated by incubating at 37 C for 30 min with 1000-collapse molar excessive oxidized glutathione (GSSG) in 50 mM CAPS buffer at pH 9.7. Untreated SOD1 was subjected to the same incubation in 50 mM CAPS buffer at pH 9.7, containing no GSSG. Following this incubation, untreated and GSSG-enriched SOD1 samples were demetalated as explained above, then brought to 100 M apo-SOD1. A 64 g aliquot was eliminated, filtered using a 0.22 m centrifugal filter, and injected onto Rabbit Polyclonal to Chk2 a Superdex 200 10/300 GL column (GE Healthcare) at 4 C equilibrated in 20 mM Tris and 150 mM NaCl at pH 7.4. Effect of Reducing Agent Treatment on Apo-SOD1 Oligomer Stability Oligomers of apo-SOD1 were prepared as explained above, and DTT was added to a final concentration of 1 1 mM to the sample and SEC operating buffer. Aliquots from your mixture of oligomers were separated by SEC as explained above immediately following the addition of DTT and after 2 h and over night incubation at space temperature. Results Formation of Metastable Soluble Oligomers by Apo-SOD1 with FALS-Linked Substitutions To identify potentially disease-relevant metastable SOD1 oligomers, we incubated apo-SOD1 at physiological pH, temp, ionic strength, and SOD1 concentration for up to one week, separating the reaction combination by size exclusion chromatography (SEC) at multiple time points. We use recombinant protein in which SOD1s native free cysteines (Cys-6 and Cys-111) are retained, as they happen to Asapiprant be demonstrated to play important tasks in oligomerization.13,14 Metal-free (apo) SOD1 is utilized in all experiments since it is widely considered to be the common precursor to misfolded and aggregated varieties.4,15,16 We analyze soluble oligomers because of their particular relevance to ALS pathology; apo-SOD1 remains soluble throughout the 1-week incubation period, as evidenced from the minimal changes in total A280 from SEC chromatograms (Number ?(Number1B,C).1B,C). WT SOD1 (Numbers ?(Numbers1B1B and ?and2B)2B) and SOD1 containing the FALS-linked G93A and G37R substitutions (Number ?(Figure2B)2B) have low propensities to form soluble oligomers less than Asapiprant these conditions, whereas SOD1 with the A4V or Asapiprant I112T substitutions shows considerable oligomerization (Figures ?(Numbers1C1C and ?and2B).2B). Analysis of SEC-separated oligomers with multiangle light scattering is definitely consistent with the presence of native-like dimers, non-native-like expanded dimers, trimers, tetramers, and hexamers (Number ?(Figure1D).1D). The presence of an expanded dimer is definitely inferred from your SEC-MALS data based on the presence of a peak eluting before the native-like dimer (indicating its larger hydrodynamic radius), Asapiprant yet having a determined molecular weight equivalent to that of the native-like dimer (reddish vs cyan curves, Number ?Number1D).1D). In the case of the aggregation-prone A4V and I112T variants, small soluble oligomers are apparent by 2 h of incubation at 37 C (Number ?(Figure1C)1C) and remain detectable throughout the 1-week incubation period. The smallest non-native oligomers (those eluting near 13 and 14.5 mL following injection onto the gel filtration column) increase in abundance for the first 8C24 h, after which their populations decrease concomitant with the appearance of higher-order species (Number ?(Number11C). Open in a separate window Number 1 Formation of metastable soluble non-native oligomers of metal-free SOD1. (A) Positions of the glutathione changes and of the FALS-linked amino acid substitutions included in the current study; residue positions are indicated by coloured spheres on the background of the WT SOD1 crystal framework (PDB Identification: 1spd). (B) SEC chromatograms displaying aggregation of 100 M.
