Abstract
Mutations in copper-zinc superoxide dismutase cause the neurodegenerative disease amyotrophic lateral sclerosis. Many of the mutant proteins have increased turnover in vivo and decreased thermal stability. Here we show that purified, metal-free superoxide dismutases are degraded in vitro by purified 20 S proteasome in the absence of ATP and without ubiquitinylation, whereas their metal-bound counterparts are not. The rate of degradation by the proteasome varied among the mutants studied, and the rate correlated with the in vivo half-life. The monomeric forms of both mutant and wild-type superoxide dismutase are particularly susceptible to degradation by the proteasome. Exposure of hydrophobic regions as a consequence of decreased thermal stability may allow the proteasome to recognize these molecules as non-native.
Highlights
Mutations in copper-zinc superoxide dismutase cause the neurodegenerative disease amyotrophic lateral sclerosis
Apo-SOD1s Are Substrates for the 20 S Proteasome but Metal-bound Forms Are Not—The as isolated SOD1 proteins are metal loaded with copper and zinc, with varying degrees of fidelity and complete
This decrease in stability of the apo wild-type dimer was sufficient to allow some digestion by the proteasome, with ϳ5% degraded during a 2-h incubation (Fig. 2)
Summary
Materials—Substrates for fluorimetric assay of proteasome activities (AAF-AMC, AMC, LLE-AMC, and LSTR-AMC), ANSA, DTPA, and lactacystin were purchased from Sigma. Disulfide Bond Formation and Scrambling—As described, we routinely assayed the susceptibility of SOD1 proteins to proteasomal degradation by the decrease in area of the peak of fulllength SOD1 in the HPLC chromatogram. A peptide of measured mass 6,363.3 corresponds to residues 91–153 with a disulfide bond between Cys111 and Cys146 We investigated whether these non-native disulfide bonds were present in the SOD1 preparations or formed during the proteasome assay or in the mass spectrometer. Metal-bound wild-type SOD1 had ϳ94% of Cys and Cys146 in disulfide linkages, whereas ϳ94% of Cys and Cys111 had free thiols. The fraction of digested SOD1 was determined from the loss of area under the intact SOD1 peak in the 210-nm chromatogram The accuracy of this assay depends on the ability of the reverse phase column to separate the intact SOD1 from all products generated by the proteasome. Results were expressed as the change in fluorescence intensity with respect to this reference
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