Fig. 1.
A specific conformation of soluble oligomers potently inhibits the mammalian 20S proteasome. a Mammalian 20S proteasomes were incubated with mixed aggregates of Aβ1–42 (5 μM), α-Syn (1 μM), Htt-53Q (0.1 μM), or an equal volume of oligomer buffer (control). Proteasome activity (linear rate of LLVY-amc hydrolysis) is represented as a percentage of activity compared to the control. b Crude aggregates from a were separated into soluble and insoluble aggregates (schematic, left) and were assayed as in a (bar graph, right). For huntingtin monomers, Htt-20Q monomers were used because pure Htt-53Q monomers could not be obtained due to rapid oligomerization. Dot blots of monomers, soluble aggregates, and insoluble aggregates from b were probed with the conformation-dependent anti-oligomer “A11” antibody (bottom right). c Soluble Aβ aggregates from b were separated by size exclusion chromatography (Abs280 nm, solid blue line). Two microliters from each fraction was evaluated for its effect on 20S proteasome chymotrypsin-like activity (bars) and probed for anti-oligomer A11 reactivity (dot blot, bottom). d Proteasome activity with up to 5 μM of Aβ oligomers (Aβ-iO) or Aβ protofibrils (Aβ-PF) from c. e Intermediate oligomers from d were pre-incubated with anti-oligomer A11 antibody (Aβ-iO + A11) or an equal volume of antibody buffer (Aβ-iO) for 30 min at 37 °C before to addition to proteasome activity assay. Final concentration of Aβ-iO in the assay was 0.25 μM (the ~IC50 as determined in d). The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass (Aβ, 4.5 kDa; α-Syn, 14 kDa; and Htt-53Q, 22 kDa). All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation