Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans
Figure 6
Hsp26 inhibits seeded assembly of Sup35 more potently than Hsp42 in a temperature-sensitive manner.
(A) NM (5 µM) was incubated at 25°C for 12 h in the presence of preformed NM fibers (5% wt/wt) plus increasing concentrations of either BSA, Hsp26, Hsp42, or Hsp26 and Hsp42 (0–24 µM). For the mixture of Hsp26 and Hsp42, a 1∶1 ratio was employed. Thus, a concentration of 2 µM on the x-axis reflects 1 µM Hsp26 and 1 µM Hsp42. For the Hsp26 alone condition, Hsp26 was pretreated at either 25°C or 45°C for 10 min. Fibrillization was measured by ThT fluorescence. Values represent means±SD (n = 3). (B) NM (5 µM) was incubated at 25°C for 12 h in the presence of preformed NM fibers (5% wt/wt) plus BSA, Hsp26, or Hsp42 (12 µM). Hsp26 was pretreated at either 25°C or 45°C for 10 min. Fibrillization was measured by determining the amount of SDS-resistant NM. Values represent means±SD (n = 3). (C) Sup35 (5 µM) was incubated at 25°C for 12 h in the presence of preformed Sup35 fibers (5% wt/wt) plus BSA, Hsp26, or Hsp42 (12 µM). Hsp26 was pretreated at either 25°C or 45°C for 10 min. Fibrillization was measured by ThT fluorescence. Values represent means±SD (n = 3). (D) Chemically denatured GDH or NM (5 µM) was incubated at 25°C for 4 h with agitation in the presence of Hsp26 (1–10 µM), which had been pretreated at either 25°C or 45°C for 10 min. GDH aggregation was assessed by turbidity and NM fibrillization by ThT fluorescence. Values represent means±SD (n = 3). (E) NM proteins (5 µM) carrying pyrene labels at the indicated single site were assembled at 25°C for 12 h in the presence of preformed NM fibers (5% wt/wt) plus either Hsp26 or Hsp42 (12 µM). Hsp26 was pretreated at either 25°C or 45°C for 10 min. The ratio of excimer to non-excimer fluorescence (I465 nm/I375 nm) was then determined as a measure of intermolecular contact formation. Soluble NM serves as a negative control.