Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans
Figure 13
sHsps promote gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40.
(A) Schematic illustrating the concept of NM-his capped NM fibers (left) or NM capped NM-his fibers (right). Cyan circles depict NM-his and white circles depict NM. (B, C) NM fibers with NM-his caps (2.5 µM monomer) were not sonicated and incubated at 25°C for 0–28 d in the absence or presence of Hsp26 (5 µM), Ssa1:Sis1 (2.5 µM each), Ssa1:Ydj1 (2.5 µM each), Sse1:Ssa1:Ydj1 (1.67 µM each), or Sse1:Ssa1:Sis1 (1.67 µM each). At the indicated times, reactions were centrifuged at 436,000 g for 30 min. The amount of SDS-soluble untagged NM (B) or NM-his (C) in the supernatant was then determined by quantitative immunoblot. Values represent means±SD (n = 3). (D, E) NM-his fibers with NM caps (2.5 µM monomer) were not sonicated and incubated at 25°C for 0–28 d in the absence or presence of Hsp26 (5 µM), Ssa1:Sis1 (2.5 µM each), Ssa1:Ydj1 (2.5 µM each), Sse1:Ssa1:Ydj1 (1.67 µM each), or Sse1:Ssa1:Sis1 (1.67 µM each). At the indicated times, reactions were centrifuged at 436,000 g for 30 min. The amount of SDS-soluble untagged NM (D) or NM-his (E) in the supernatant was then determined by quantitative immunoblot. Values represent means±SD (n = 3). (F, G) NM-his fibers with NM caps (2.5 µM monomer) were sonicated and incubated at 25°C for 0–28 d in the absence or presence of Hsp26 (5 µM), Ssa1:Sis1 (2.5 µM each), Ssa1:Ydj1 (2.5 µM each), Sse1:Ssa1:Ydj1 (1.67 µM each), or Sse1:Ssa1:Sis1 (1.67 µM each). At the indicated times, reactions were centrifuged at 436,000 g for 30 min. The amount of SDS-soluble untagged NM (F) or NM-his (G) in the supernatant was then determined by quantitative immunoblot. Values represent means±SD (n = 3). (H, I) NM fibers (2.5 µM) were sonicated and then incubated for 1 h at 25°C with buffer, Hsp26, or Hsp42 (10 µM). (H) Sse1:Ssa1:Sis1 (1.67 µM each) or (I) Sse1:Ssa1:Ydj1 (1.67 µM each) was then added and fibers were incubated for 0–28 d at 25°C. At the indicated times, reactions were centrifuged at 436,000 g for 30 min. The amount of SDS-soluble NM in the supernatant was then determined by quantitative immunoblot. Values represent means±SD (n = 3).