Pure Hsp110, Hsp70 and Hsp40 drive protein disaggregation.

<p>(<b>A, B</b>) Urea-denatured luciferase aggregates (50 nM) (<b>A</b>) or heat-denatured GFP aggregates (0.45 µM) (<b>B</b>) were incubated for 30 min (blue bars) or 6 h (red bars) at 37°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsp104 (1 µM), Ssa1 (1 µM), Ydj1 (1 µM), RLC (10 mg/ml), SHC (10 mg/ml), Hsc70 (1 µM), Hdj1 (1 µM), Hdj2 (1 µM), Hsp70 (1 µM) and Apg-2 (1 µM). In the indicated reactions, ATP was omitted or replaced with AMP-PNP. Alternatively, ATP was included but the ATP-regeneration system was replaced with apyrase. The small molecule inhibitor VER 155008 (100 µM) was included in the indicated reactions. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (<b>A</b>). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (<b>B</b>). Values represent means ± SEM (n = 3).</p

Mammalian cytosol contains an ATPase-dependent disaggregation machinery.

<p>(<b>A, B</b>) Urea-denatured luciferase aggregates (50 nM) (<b>A, C</b>) or heat-denatured GFP aggregates (0.45 µM) (<b>B, D</b>) were incubated for 30 min (blue bars), 1 h (green bars), 4 h (black bars), or 6 h (red bars) at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsp104 (1 µM), Ssa1 (1 µM), Sis1 (1 µM), RLC (10 mg/ml) (<b>A, B</b>), or SHC (10 mg/ml) (<b>C, D</b>). In the indicated reactions, ATP was omitted or replaced with AMP-PNP. Alternatively, ATP was included but the ATP-regeneration system was replaced with apyrase. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (<b>A, C</b>). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (<b>B, D</b>). Values represent means ± SEM (n = 3).</p

Hsp110, Hsp70 and Hsp40 are key components of the mammalian disaggregation machinery.

<p>(<b>A, B</b>) Immunoblots showing depletion of either p97, Hsp70, Hsc70, Hdj1, Hdj2, Hsp105 or Apg-2 from either RLC (<b>A</b>) or SHC (<b>B</b>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026319#s3" target="_blank">Materials and Methods</a>. 20 µg of cytosol was fractionated by SDS-PAGE and processed for immunoblot. (<b>C–F</b>) Urea-denatured luciferase aggregates (50 nM) (<b>C, D</b>) or heat-denatured GFP aggregates (0.45 µM) (<b>E, F</b>) were incubated for 6 h in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated cytosol (10 mg/ml), mock-depleted cytosol (10 mg/ml) or depleted cytosol (10 mg/ml). In the indicated reactions, the small molecule p97 inhibitor, DBeQ (100 µM), or Hsp70 inhibitor, VER 155008 (100 µM), were included. In the indicated reactions, Hsc70-depleted cytosol was supplemented with Hsc70, Hdj1-depleted cytosol was supplemented with Hdj1 and Apg-2-depleted cytosol was supplemented with Apg-2 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026319#s3" target="_blank">Materials and Methods</a>. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (<b>C, D</b>). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (<b>E, F</b>). Values represent means ± SEM (n = 3).</p

Hsp110 NEF activity, ATPase activity and substrate-binding activity are essential for protein disaggregation.

<p>(<b>A, B</b>) Urea-denatured luciferase aggregates (50 nM) (A) or heat-denatured GFP aggregates (0.45 µM) (B) were incubated for 30 min (blue bars), 1 h (green bars), 4 h (black bars), or 6 h (red bars) at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsp104 (1 µM), Ssa1 (1 µM), Sis1 (1 µM) and Sse1 (1 µM). In the indicated reactions, ATP was either omitted or replaced with AMP-PNP. Alternatively, ATP was included but the ATP-regeneration system was replaced with apyrase. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (<b>A</b>). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (B). Values represent means ± SEM (n = 3). (<b>C, D</b>) Urea-denatured luciferase aggregates (50 nM) (<b>C</b>) or heat-denatured GFP aggregates (0.45 µM) (<b>D</b>) were incubated for 30 min (blue bars), 1 h (green bars), 4 h (black bars), or 6 h (red bars) at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Ssa1 (1 µM), Sis1 (1 µM), Sse1 (1 µM), Sse1^N572Y:E575A (1 µM, a NEF-defective Sse1 mutant), Ssa1^A300E (1 µM, an Ssa1 mutant unable to interact with Sse1), Fes1 (1 µM, a Ssa1 NEF), Snl1ΔN (1 µM, a Ssa1 NEF), Sse1^K69M (1 µM, an ATPase-dead Sse1 mutant), Ssa1^K69Q (1 µM, an ATPase-dead Ssa1 mutant), Sse1^L433A:N434P (1 µM, a substrate-binding defective Sse1 mutant), Sse1^F439L:M441A (1 µM, a substrate-binding defective Sse1 mutant), Ssa1^L483W (1 µM, a substrate-binding defective Ssa1 mutant), Sis1^H34Q (1 µM, a Sis1 mutant unable to stimulate Ssa1 ATPase activity), Sis1^1–68 (1 µM, the J domain of Sis1), Sis1^1–121 (1 µM, the J and G/F-domains of Sis1) and Ydj1 (1 µM, an Hsp40). Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (C). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (D). Values represent means ± SEM (n = 3).</p

Pure Hsp110, Hsp70 and Hsp40 enhance Hsp104 disaggregase activity against Sup35 prions and α-syn amyloid fibers.

<p>(<b>A, B</b>) Preformed Sup35 prions (2 µM monomer) were incubated for 6 h at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Ssa1 (2 µM), Sis1 (2 µM), Sse1 (2 µM) (each individual combination is an individual category of the x-axis) in the absence (blue bars) or presence of Hsp104 (0.5 µM, green bars; or 2 µM, black bars). For the 10x(Sse1+Ssa1+Sis1) condition the concentration of Ssa1, Sis1 and Sse1 was increased to 20 µM. Sup35 prion disassembly was assessed by the amount of SDS-resistant Sup35 (<b>A</b>) or by ThT fluorescence (<b>B</b>). Values represent means ± SEM (n = 3). (<b>C, D</b>) Preformed amyloid fibers composed of α-syn (0.5 µM monomer) were incubated for 6 h at 37°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsc70 (10 µM), Hdj1 (10 µM), Hsp70 (10 µM) and Apg-2 (10 µM) (each individual combination is an individual category of the x-axis) in the absence (blue bars) or presence of Hsp104 (2.5 µM, green bars; or 10 µM, black bars). Fiber integrity was then determined by sedimentation analysis (<b>C</b>) or by ThT fluorescence (<b>D</b>). Values represent means ± SEM (n = 3).</p

sHsps promote gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40.

<p>(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 <i>not</i> 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 (<i>n</i> = 3). (D, E) NM-his fibers with NM caps (2.5 µM monomer) were <i>not</i> 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 (<i>n</i> = 3). (F, G) NM-his fibers with NM caps (2.5 µM monomer) <i>were</i> 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 (<i>n</i> = 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 (<i>n</i> = 3).</p

Hsp26 and Hsp42 synergize to inhibit spontaneous Sup35 prionogenesis.

<p>(A, B) NM (5 µM) was incubated at 25°C with agitation for 6 h in the presence of increasing concentrations of BSA, Hsp26, Hsp42, or Hsp26 and Hsp42 (0–5 µM). For the mixture of Hsp26 and Hsp42, a 1∶1 ratio was employed. Thus, a concentration of 2 µM on the <i>x</i>-axis reflects 1 µM Hsp26 and 1 µM Hsp42. Fibrillization was measured by Thioflavin-T (ThT) fluorescence (A) or by determining the amount of SDS-resistant NM (B). Values represent means±SD (<i>n</i> = 3). (C) NM (5 µM) was assembled at 25°C with agitation for 6 h in the absence or presence of Hsp26 (0.6 µM or 3 µM), Hsp42 (0.6 µM or 3 µM), or Hsp26 and Hsp42 (0.3 µM or 1.5 µM of each). Reaction products were concentrated and transformed into [<i>psi</i>⁻] cells. No NM and soluble NM served as negative controls. The proportion of [<i>PSI</i>⁺] colonies was then determined. Values represent means±SD (<i>n</i> = 3). (D) Sup35 (5 µM) was incubated at 25°C with agitation for 6 h in the presence of increasing concentrations of BSA, Hsp26, Hsp42, or Hsp26 and Hsp42 (0–5 µM). For the mixture of Hsp26 and Hsp42, a 1∶1 ratio was employed. Thus, a concentration of 2 µM on the <i>x</i>-axis reflects 1 µM Hsp26 and 1 µM Hsp42. Fibrillization was measured by ThT fluorescence. Values represent means±SD (<i>n</i> = 3).</p

Hsp26 inhibits seeded assembly of Sup35 more potently than Hsp42 in a temperature-sensitive manner.

<p>(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 <i>x</i>-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 (<i>n</i> = 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 (<i>n</i> = 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 (<i>n</i> = 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 (<i>n</i> = 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 (I_{465 nm}/I_{375 nm}) was then determined as a measure of intermolecular contact formation. Soluble NM serves as a negative control.</p

Hsp26 or Hsp42 binding destabilizes NM fibers.

<p>(A) NM fibers (5 µM NM monomer) were incubated for 60 min at 25°C without or with Hsp26 (10 µM), Hsp42 (10 µM), or Ssa1:Sis1 (10 µM of each) in the presence of ATP (5 mM). The stability of the various NM fibers was then determined by SDS–PAGE and quantitative immunoblot. The amount of SDS-soluble NM, which reflects susceptibility of NM fibers to thermal solubilization, was plotted against temperature and fitted to a sigmoidal function. Values represent means±SD (<i>n</i> = 3). (B) NM proteins (5 µM) carrying pyrene labels at the indicated single site were assembled at 25°C with agitation for 12 h. Assembled NM fibers (5 µM NM monomer) were then incubated for 60 min at 25°C without or with buffer, Hsp26, Hsp42, or Ssa1:Sis1 (10 µM) in the presence of ATP (5 mM). The ratio of excimer to non-excimer fluorescence (I_{465 nm}/I_{375 nm}) was then determined.</p

Human HspB5 promotes gradual depolymerization of α-syn fibers by human Hsp110, Hsp70, and Hsp40.

<p>(A, B) α-Syn fibers (2.5 µM monomer) were sonicated and then incubated for 0–30 d at 25°C with either buffer, Hsc70 (10 µM), Hdj1 (10 µM), Apg-2 (10 µM), HspB5 (10 µM), Hsc70:Hdj1 (5 µM of each), Hsc70:Apg-2 (5 µM of each), Hsc70:HspB5 (5 µM of each), Hdj1:Apg-2 (5 µM of each), Hdj1:HspB5 (5 µM of each), Apg-2:HspB5 (5 µM of each), Hsc70:Hdj1:Apg-2 (3.3 µM of each), Hsc70:Apg-2:HspB5, Hsc70:Hdj1:HspB5, Hdj1:Apg-2:HspB5 (3.3 µM of each), or Hsc70:Hdj1:Apg-2:HspB5 (2.5 µM of each). At the indicated times, reactions were centrifuged at 436,000 g for 30 min (A). The amount of α-syn in the supernatant was then determined by quantitative immunoblot (A). Alternatively, fiber integrity was assessed by ThT fluorescence (B). Values represent means±SD (<i>n</i> = 3). (C) α-Syn fibers with his-α-syn caps (2.5 µM monomer) were <i>not</i> sonicated and incubated at 25°C for 0–30 d in the presence of Hsc70:Hdj1:Apg-2:HspB5 (2.5 µM of each). At the indicated times, reactions were centrifuged at 436,000 g for 30 min. The amount of his-α-syn (blue) or untagged α-syn (red) in the supernatant was then determined by quantitative immunoblot. Values represent means±SD (<i>n</i> = 3). (D) Preformed amyloid fibers composed of α-syn (0.5 µM monomer) were incubated for 6 h at 37°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of HspB5 (10 µM), Hsc70 (10 µM), Hdj1 (10 µM), and Apg-2 (10 µM) in the absence (blue bars) or presence of Hsp104 (2.5 µM, red bars; or 10 µM, green bars). Fiber integrity was then determined by sedimentation analysis and quantitative immunoblot. Values represent means±SD (<i>n</i> = 3).</p