Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase

Abstract

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.

Keywords: atomic force microscopy; cryo-electron tomography; disaggregation; molecular chaperones.

Figure 1. Frames from atomic force microscopy (AFM) movies of αSyn fibre disaggregation by Hsc70/DNAJB1/Apg2

1. An example video series showing the disaggregation of an αSyn fibre. The time stamp is in hours:minutes:seconds, with the chaperone/ATP mixture added at time 0:00:00. Scale bar, 250 nm. The height (colour) scale is 0 (black) to 26 nm (white), see inset at bottom right, where 0 nm refers to the passivated mica substrate.

2. Video frames showing key stages of the disaggregation process. Time stamps and scale bar as in (A).

3. Frames from AFM movie series that show the polar disassembly of αSyn fibres. The white arrows indicate the direction of depolymerisation. Time stamps as above and scale bar 200 nm. The videos are provided as expanded view movies EV1 and EV2.

Data information: Green arrows indicate fragmentation sites. White arrows indicate the direction of depolymerisation along fibres.

Figure EV1. Disaggregation seen in AFM movies requires ATP

Snapshots of an AFM time‐series showing αSyn fibres before addition of chaperones (left panels) and after incubation with chaperones for 2 h (right panels) in the presence (top panels) and absence (bottom panels) of ATP. The top panels show the same fibre as in Fig 1A and Movie EV1. The scale bars represent 250 nm.

Figure 2. Time course of depolymerisation and morphological changes

1. Example segment of an αSyn fibre depolymerised in a rapid “burst”, which removed 111 nm of this fibre (indicated with white marker) within two to three successive video frames (i.e. within 3–4 min). Height (colour) scale 26 nm, as in Fig 1. Scale bar: 50 nm.

2. A kymograph showing the disaggregation of a single αSyn fibre: (i) The first frame of an atomic force microscopy (AFM) video series of disaggregation. Scale bar: 50 nm. (ii) Kymograph showing a height profile that is aligned to the fibre shown in A (white dotted line) as a function of time (horizontal axis of kymograph). Zero minutes is defined as the first frame recorded after the chaperones were injected into the solution. The white arrows indicate depolymerisation events. (iii) The same, now largely disaggregated, αSyn fibre as shown in A, at the end of the movie.

3. Lengths of individual fibres plotted as a function of time through disaggregation reactions as imaged by AFM. Traces are shown for a subset of six fibres, each in a different colour, from four independent experiments.

4. Disaggregation videos that show that a local height increase of αSyn fibres precedes depolymerisation. Zero time here refers to the latest frame before changes to the fibre were observed. Height (colour) scale: 21 nm, see Fig 1. Scale bar: 50 nm.

5. Height profiles along the dashed lines in D showing the changes to αSyn fibres during disaggregation. The green trace was taken immediately before the increase in fibre height, and shows the undulations in fibre height due to its helical structure. The blue trace was recorded approximately 2 min after the change in fibre height (green trace), and it shows this height increase at the left‐hand end of the fibre. After about 17 min from the initial height change, the red trace shows the removal of the fibre over the same region that was previously elevated.

Figure EV2. The helical periodicity does not account for the bursts of disassembly

1. The chaperone binding sites are on the flexible αSyn termini, which are expected to follow the helical path of the fibre structure.

2. We examined whether local attachment to the substrate might arrest disaggregation and account for the burst‐like depolymerisation events observed by AFM. No such relationship is evident between the helical repeat and the positions where disaggregation is arrested.

3. The length of the first disaggregated segment is not less on average than that of subsequently disaggregated segments.

Figure EV3. Alternative explanations of the fibre height increase seen by AFM

1. A–C

   The small and uniform height increase preceding a depolymerisation burst does not resemble local detachment from the mica. Local detachment from the mica would not result in a uniform height increase as in A, but would cause increasing mobility of the segment with distance from the detachment point, and a corresponding loss of imaging resolution (B). Dense chaperone binding over the elevated region (C) remains the only plausible explanation accounting for the AFM observations.

Figure 3. Cryo EM images and tomograms of fibre–chaperone complexes

1. A, B

   Representative cryo EM images of αSyn fibres alone (A), αSyn fibres incubated with Hsc70/DNAJB1/Apg2/ATP (B). White arrowheads show sites of decoration.

2. C

   Tomogram section of fibres incubated with Hsc70, DNAJB1 and ATP. Small clusters are interspersed with sparse decoration.

3. D

   Tomogram section of fibres incubated with Hsc70, DNAJB1, Apg2 and ATP, showing extended stretches of densely clustered chaperones. The two‐protofilament structure of the fibres is evident, but the flexible αSyn N‐ and C‐terminal tails that contain the chaperone binding sites are not discernible.

Data information: Scale bars, 30 nm.

Figure 4. Hsc70 recruitment is greatly stimulated by Apg2

1. A

   Binding assay for Hsc70 binding in the presence of WT DNAJB1: control of DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70/Apg2, control of DNAJB1/Hsc70/Apg2. The ‘*’ highlights the condition where an increase of Hsc70 in the bound fraction is observed. P, pellet; S, supernatant. The amount of Hsc70 recruited to the fibres is shown in the pellet fractions.

2. B

   Binding assay for Hsc70 binding in the presence of the truncated ΔJ‐DNAJB1, lacking the J domain. The gel layout is similar to the previous one with ΔJ‐DNAJB1 replacing WT DNAJB1. Both Apg2 and J domain are required for enhancement of Hsc70 binding.

3. C

   Histogram of Hsc70 bound fraction in each sample (N = 3 independent experiments, WT: WT DNAJB1, ΔJ: ΔJ‐DNAJB1 mutant, ‐Apg2: condition with fibres + DNAJB1/Hsc70, +Apg2: condition with fibres + DNAJB1/Hsc70/Apg2). A 2‐way ANOVA was performed with Tukey’s multiple comparisons test (**P = 0.003; ****P < 0.0001). Mean Hsc70 binding values with SD are shown.

4. D

   Total internal reflection fluorescence microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2. Scale bar, 1 µm.

5. E, F

   Quantitation of fluorescence intensity of Hsc70 (E) and αSyn (F) in the two conditions (+/− Apg2, N = 3 independent experiments, n = 65 fibres). Mean Hsc70 and αSyn fluorescence intensities with SD are shown.

6. G

   Quantitation of differences in Hsc70 binding between the two ends of the fibres, normalised to the local αSyn intensity, in the absence and presence of Apg2 (N = 3 independent experiments, n = 54 fibres). Mean Hsc70 fluorescence intensities with SD are shown.

Data information: (E–G) For each plot, data were tested for normality by performing a Shapiro–Wilk test. As the data did not display a normal distribution, they were analysed using a two‐tailed Mann–Whitney test (****P < 0.0001, ns: not significant).

Figure EV4. Negative stain EM images showing that chaperone recruitment to fibres did not occur when fibres were incubated with Hsc70/ΔJ‐DNAJB1/Apg2/ATP

1. A–C

   In negative stain images, chaperone binding appeared as a pronounced increase in fibre thickness, visible when comparing images of αSyn samples alone to those incubated with Hsc70/DNAJB1/Apg2. (A) αSyn fibres alone; (B) fibres + Hsc70/DNAJB1/Apg2/ATP; (C) fibres + Hsc70/ΔJ‐DNAJB1/Apg2/ATP. Scale bar, 50 nm.

Figure 5. Dependence of Hsc70 recruitment and disaggregation activity on Apg2 concentration

1. A, B

   Binding assay with different dilutions of Apg2 incubated with DNAJB1/Hsc70 and (A) with αSyn fibres or (B) without αSyn fibres (control). 5‐mM ATP was present in the buffer throughout.

2. C

   Plot showing Hsc70 binding as a function of Apg2:Hsc70 molar ratio (N = 3 independent experiments). The action of Apg2 in recruiting Hsc70 to the fibres saturates at a molar ratio of Apg2:Hsc70 of 1:10. The curve indicates the mean Hsc70 binding values.

3. D

   Disaggregation activity measured by Thioflavin T fluorescence shows the same dependence on Apg2 as binding assays. High Apg2 (1:2 molar ratio) makes disaggregation less efficient, possibly by prematurely releasing Hsc70.

4. E

   Quantification of the disaggregation efficiency as a function of Apg2:Hsc70 molar ratio (N = 3 independent experiments). The disaggregation efficiency was determined by taking the values from the final hour of the assay curves in (D). The curve indicates the mean efficiency values.

5. F

   Plot showing the linear correlation between disaggregation activity and Hsc70 binding. Including the data point without Apg2 did not change the correlation coefficient, which remained at 0.967. In contrast, including the highest Apg2 concentration (Apg2:Hsc70 molar ratio of 1:2) reduced the correlation coefficient to 0.904. Mean values with SD are shown.

Figure 6. Polarity of the Hsc70 binding site in the ordered part of the fibre structure

The polar structure of the fibre is shown with electrostatic surface colouring, showing a crucial Hsc70 binding site on αSyn with a more favourable exposure of positive charge (blue) on one surface than on the other. The Hsc70 binding site 37–43 is outlined in green on one protofilament. This figure shows PDB 6cu7 (B. Li et al, ; Data ref: B Li et al, 2018) which has a very similar fold to fibres used in much of our analysis.

Figure 7. Proposed model of αSyn fibre depolymerisation

Apg2 catalyses the assembly of densely packed clusters of Hsc70, priming the rapid bursts of disaggregation from one end of the fibre.

Figure EV5. Disaggregation activity measured by Thioflavin T fluorescence

Labelling T111C Hsc70 mutant with the 488 maleimide dye does not affect the efficiency of the disaggregation.