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Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans

Figure 3

Hsp26 and Hsp42 inhibit de novo Sup35 prionogenesis by distinct mechanisms.

(A) NM (5 µM) was rotated for 5 min (80 rpm) in the absence or presence of Hsp26 (3 µM), Hsp42 (3 µM), or Ssa1 plus Ydj1 (3 µM). Oligomeric NM was recovered by centrifugation at 436,000 g for 30 min, resolved by SDS-PAGE, Coomassie stained, and the amount in the pellet fraction determined. Values represent means±SD (n = 3). (B) Fluorescence of NM-N21C-, Q38C-, G96C-, or Y106C-acrylodan (5 µM) after 15 min at 25°C in the absence or presence of BSA (3 µM), Hsp26 (3 µM), Hsp42 (3 µM), or Hsp26 (1.5 µM) and Hsp42 (1.5 µM). Values represent means±SD (n = 3). (C, D) NM (5 µM) was incubated at 25°C with agitation for 10 min at which point (arrow) buffer, Hsp26 (3 µM), or Hsp42 (3 µM) was added. The reaction was then continued at 25°C with agitation to 150 min. At the indicated times, the amount of A11-reactive species present was determined (C) or the amount of ThT-reactive species was determined (D). Datasets representative of three replicates are shown. (E) Electron microscopy of NM assembly at 25°C with agitation for 6 h in the absence or presence of Hsp26 (3 µM), Hsp42 (3 µM), or Hsp26 (1.5 µM) and Hsp42 (1.5 µM). Note the presence of small fibers in the presence of Hsp26 (arrow), the accumulation of oligomers in the presence of Hsp42 or Hsp26 and Hsp42. Bar, 0.5 µm. (F) NM cysteine variants were either left uncrosslinked or crosslinked under denaturing conditions with a flexible 11 Å BMB crosslink at position 25 or 31. The indicated NM protein (5 µM) was then assembled with agitation at 25°C in the absence or presence of BSA (3 µM), Hsp26 (3 µM), Hsp42 (3 µM), or Hsp26 and Hsp42 (1.5 µM of each). Fibrillization was measured by ThT fluorescence. Values represent means±SD (n = 3).

Figure 3

doi: https://doi.org/10.1371/journal.pbio.1001346.g003