Figure 6. α-Syn-specific Hsp104 variants have distinct biochemical properties.

(A) ATPase activity of Hsp104 variants. Values represent means±SEM (n=4). (B) HAP variants (0.167μM hexamer) plus ClpP (21μM monomer) were incubated with FITC-casein, and FITC-casein degradation was measured by fluorescence. Negative controls FITC-casein alone (black) and ClpP alone control (dark brown) were included. Values were normalized to HAP plus ClpP at 60 minutes and represent means±SEM (n=3–6). (C) RepA_1-25-GFP (0.7μM) was incubated with Hsp104 variants (2.1μM hexamer) plus GroEL^trap (2.5μM tetradecamer), and RepA_1-25-GFP unfolding was measured by fluorescence. Negative control of RepA_1-25-GFP alone included. A representative trial is shown (n=3). (D) Hsp104 variants alone (1μM hexamer, solid bars) or with chaperone pairs Hsc70 and Hdj2 (0.167μM each, checkered bars), or Hsp72 and Hdj1 (0.167μM each, dotted bars), were incubated with urea-denatured luciferase aggregates. Reactivation activity was measured by luminescence. Negative control of luciferase aggregates alone included. Values represent means±SEM (n=2–8). (E) α-Syn fibrils (3µM monomer) were incubated without or with Hsp104, Hsp104^A503S, Hsp104^K358D, Hsp104^K358D:Y257T or Hsp104^K358D:Y662M (3µM) plus Hsc70 (3µM), Hdj1 (3µM), ATP (20 mM) and an ATP regeneration system (20mM creatine phosphate and 0.5µM creatine kinase) for 2h at 30°C. Disaggregation was assessed by Thioflavin-T (ThT) fluorescence. Values represent means±SEM (n=3). (F) TDP-43 fibrils (3µM monomer) were incubated without or with Hsp104, Hsp104^A503S, Hsp104^K358D, Hsp104^K358D:Y257T or Hsp104^K358D:Y662M (3µM) plus Hsc70 (3µM), Hdj1 (3µM), ATP (20mM) and an ATP regeneration system (20mM creatine phosphate and 0.5µM creatine kinase) for 2h at 30°C. Disaggregation was assessed by turbidity (absorbance at 350nm). Values represent means±SEM (n=3). (G) Soluble α-syn oligomers (0.5µM monomer) were incubated without or with Hsp104, Hsp104^A503S, Hsp104^K358D, Hsp104^K358D:Y257L, Hsp104^K358D:Y257T or Hsp104^K358D:Y662M (1µM) plus ATP (20mM) and an ATP regeneration system (20mM creatine phosphate and 0.5µM creatine kinase) for 1h at 37°C. Reactions were then fractionated through a Microcon YM-100 (100-kDa molecular weight cut off) filter. The amount of α-syn in the filtrate fraction was then determined. Values represent means±SEM (n=3).

See also Figure S6.