Regulation of α-synuclein by chaperones in mammalian cells

Abstract

Neurodegeneration in patients with Parkinson’s disease is correlated with the occurrence of Lewy bodies—intracellular inclusions that contain aggregates of the intrinsically disordered protein α-synuclein1. The aggregation propensity of α-synuclein in cells is modulated by specific factors that include post-translational modifications2,3, Abelson-kinase-mediated phosphorylation4,5 and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear6,7,8. Here we systematically characterize the interaction of molecular chaperones with α-synuclein in vitro as well as in cells at the atomic level. We find that six highly divergent molecular chaperones commonly recognize a canonical motif in α-synuclein, consisting of the N terminus and a segment around Tyr39, and hinder the aggregation of α-synuclein. NMR experiments9 in cells show that the same transient interaction pattern is preserved inside living mammalian cells. Specific inhibition of the interactions between α-synuclein and the chaperone HSC70 and members of the HSP90 family, including HSP90β, results in transient membrane binding and triggers a remarkable re-localization of α-synuclein to the mitochondria and concomitant formation of aggregates. Phosphorylation of α-synuclein at Tyr39 directly impairs the interaction of α-synuclein with chaperones, thus providing a functional explanation for the role of Abelson kinase in Parkinson’s disease. Our results establish a master regulatory mechanism of α-synuclein function and aggregation in mammalian cells, extending the functional repertoire of molecular chaperones and highlighting new perspectives for therapeutic interventions for Parkinson’s disease.

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Fig. 1: Molecular chaperones prevent aggregation through the interaction with the N terminus of α-synuclein.
Fig. 2: The interaction between α-synuclein and chaperones is dominant in living cells.
Fig. 3: Co-localization of α-synuclein and cellular organelles assessed using immunofluorescence.
Fig. 4: Effect of post-translational modifications on the chaperone–α-synuclein interaction.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank S. Grzesiek, D. Otzen, M. Goedert, B. Bukau, D. P. Mulvihill and D. Kahne for providing plasmids; T. Maier and M. Plodinec for providing mammalian cell lines; E. Stuttfeld and D. Asgeirsson for technical help with cell-culture experiments; V. Juvin for help with graphic design; and the Swedish NMR Centre of the University of Gothenburg for spectrometer time. This work was supported by the Swiss National Science Foundation (PP00P3_128419 to S.H., Ambizione Fellowship PZ00P3_148238 to B.M.B. and Marie Heim-Vögtlin PMPDP3_164425 to S.C.) as well as the European Research Council (FP7 contract MOMP 281764 to S.H.). B.M.B. also acknowledges funding from the Swedish Research Council and the Knut and Alice Wallenberg Foundation through a Wallenberg Academy Fellowship as well as through the Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden. S.G.D.R. was supported by a ZonMW TOP grant (91215084) and Marie-Curie Actions of the 7th Framework programme of the EU (608180).

Author information

B.M.B. expressed and purified chaperones, and performed NMR experiments. B.M.B. and S.C. expressed and purified α-synuclein variants with help of P.K. E.E.A. and D.Š. supported protein purification of chaperones as well as α-synuclein variants. J.A.G. prepared and performed in-cell NMR experiments as well as chaperone knockdown experiments and immunofluorescence experiments. S.C. and D.G. performed the aggregation assays. I.M.-B. performed cell-culture experiments, prepared lipid vesicles, performed and analysed mass spectrometry experiments together with T.B. and A.S. A.M. performed model calculations. M.W. and S.G.D.R. provided purified HSP90β for interaction studies. B.M.B., S.C., R.R. and S.H. designed the study, analysed the data and wrote the manuscript with input from all co-authors.

Correspondence to Björn M. Burmann or Roland Riek or Sebastian Hiller.

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Competing interests

The authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Gary J. Pielak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Interaction between α-synuclein and bacterial chaperones.

ac, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-15N]-α-synuclein in the absence (grey) and presence (orange, red or dark red) of 500 µM chaperones. The sequence-specific assignments for significantly affected resonances are indicated. d, Residue-resolved chemical-shift perturbations of α-synuclein caused by the addition of two equivalents of SecB tetramer (yellow), Trigger Factor dimer (orange), Skp trimer (red) or SurA dimer (dark red). Broken lines indicate a significance level of two s.d. from the mean. e, Temperature dependence of the α-synuclein interaction with either SecB (yellow) or Skp (red) monitored by residue-resolved intensity ratios (Irel = I/I0) of 13C-direct-detected two-dimensional [15N, 13C]-NMR spectra. The intensity ratios of two-dimensional [15N, 1H]-NMR spectra at 281 K (Fig. 1c) are shown as an outline (grey). f, g, Overlay of two-dimensional [13C, 15N]-NMR spectra of 500 µM [U-13C, 15N]-α-synuclein in the absence (grey) and presence of 1 mM of SecB tetramer (f; yellow) or 1 mM of Skp trimer (g; red). Experiments were performed at 281 K and 310 K as indicated. The sequence-specific resonance assignment is shown. Experiments in ac, f, g were done in duplicates, with similar results. Source data

Extended Data Fig. 2 Chaperones Skp and Trigger Factor bind α-synuclein at their native client sites.

a, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-2H, 15N]-Skp in the absence (grey) and presence (red) of 750 µM α-synuclein. b, Residue-resolved NMR signal intensity ratios (Irel = I/I0) of Skp (250 µM) in the presence of three equivalents of α-synuclein measured at 310 K. The thin dashed lines indicate a significance level of one s.d. from the mean. The solid line represents an intensity ratio of 1. c, α-Synuclein induced intensity changes plotted on the Skp crystal structure (RCSB Protein Data Bank code (PDB) 1SG2)32 and previously reported effects upon binding of its native client OmpX10. A decrease in the signal intensity of more than one s.d. is highlighted in blue, whereas an increase in signal intensity is highlighted in red. d, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-2H, 15N]-Skp in the absence (grey) and presence (blue) of 500 µM BSA. e, Residue-resolved NMR signal intensity ratios (Irel = I/I0) of Skp (250 µM) in the presence of two equivalents of BSA measured at 310 K. The solid line represents an intensity ratio of 1. f, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-2H, 15N]-TF(∆RBD), a monomeric Trigger Factor (TF) variant that lacks its ribosome-binding and main dimerization domain (RBD), in the absence (grey) and presence (orange) of 750 µM α-synuclein. g, Residue-resolved NMR signal intensity ratios (Irel = I/I0) of 250 µM TF(∆RBD) in the presence of three equivalents of α-synuclein measured at 298 K. The thin broken lines indicate a significance level of one s.d. from the mean. The thick line represents an intensity quotient of 1. h, Residue-resolved combined chemical-shift differences of the amide moieties. The broken line indicates a significance level of two s.d. from the mean. i, Significant chemical-shift changes (green) and intensity decrease (blue) plotted on the Trigger Factor structure (PDB 1W26)33. Comparison with the published Trigger Factor interaction sites of PhoA (orange)34. j, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-2H, 15N]-TF(∆RBD) in the absence (grey) and presence (blue) of 500 µM BSA. k, Residue-resolved NMR signal intensity ratios (Irel = I/I0) of TF(∆RBD) (250 µM) in the presence of two equivalents of BSA measured at 298 K. The solid line represents an intensity ratio of 1. Experiments with α-synuclein (a, f) were done as duplicates yielding similar results, whereas control experiments with BSA (d, j) were performed once. Source data

Extended Data Fig. 3 Interaction between α-synuclein and mammalian proteins.

a, Overlay of two-dimensional [15N, 1H]-NMR spectra of 25 µM [U-15N]-α-synuclein in the absence (grey) and presence (light blue) of 50 µM inhibited HSP90β dimer. Measured in NMR buffer plus 5 mM MgCl2, 5 mM ATP, 1 µM radicicol and 1 µM geldanamycin. b, Overlay of two-dimensional [15N, 1H]-NMR spectra of 100 µM [U-15N]-α-synuclein in the absence (grey) and presence (light blue) of 200 µM HSC70. c, Overlay of two-dimensional [15N, 1H]-NMR spectra of 100 µM [U-15N]-α-synuclein in the absence (grey) and presence (light blue) of 200 µM HSC70ADP. Measured in NMR buffer plus 5 mM MgCl2 and 5 mM ADP. d, Overlay of two-dimensional [15N, 1H]-NMR spectra of 100 µM [U-15N]-α-synuclein in the absence (grey) and presence (light blue) of 200 µM HSC70ATP. Measured in NMR buffer plus 5 mM MgCl2 and 5 mM ATP. e, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-15N]-α-synuclein in the absence (grey) and presence (blue) of 500 µM (33 mg ml−1) BSA. f, Overlay of two-dimensional [15N, 1H]-NMR spectra of 250 µM [U-15N]-α-synuclein in the absence (grey) and presence (dark blue) of 500 µM of ubiquitin. g, Residue-resolved combined chemical-shift perturbations of amide moieties upon addition of HSP90β (cyan), inhibited HSP90β (light cyan), HSC70 (light blue), HSC70ADP (light blue), HSC70ATP (light blue), BSA (blue) and ubiquitin (dark blue). Broken lines indicate a significance level of two s.d. from the mean. h, Residue-resolved backbone amide NMR signal attenuation (Irel = I/I0) of α-synuclein caused by the addition of two equivalents of inhibited HSP90β (light cyan), HSC70 (light blue), HSC70ATP (light blue) and BSA (blue). i, Residue-resolved NMR signal attenuation (Irel = I/I0) of 100 µM [U-15N]-α-synuclein upon addition of increasing BSA concentrations (50–250 mg ml−1). j, Residue-resolved NMR signal attenuation (Irel = I/I0) of 50 µM [U-15N]-α-synuclein upon addition of increasing ubiquitin concentrations (25–125 mg ml−1). k, Local hydrophobicity of α-synuclein plotted against the amino acid sequence. ΔF are the free energies of transfer of the individual amino acids from an aqueous solution to its surface35. Hydrophobicity corresponds to negative ΔF values. An exponentially weighted seven-window average was applied to the raw data, with the edges contributing 50%. The red line indicates the average value of 1.5 s.d. from the mean, the chosen threshold for the identification of the most hydrophilic segments. l, Sequence-dependent DnaK score for α-synuclein derived from a computational DnaK prediction algorithm36. Regions of the primary sequence with scores less than −5 (red line) are predicted to bind DnaK, a bacterial homologue of HSC70. Experiments in af were done in duplicates with similar results. Source data

Extended Data Fig. 4 Kinetic analysis of the interaction of the chaperones with α-synuclein variants.

ac, Kinetic analysis by bio-layer interferometry of biotinylated Skp (a), SecB (b) and HSC70ADP (c) to different α-synuclein variants (α-synuclein (top), acetyl-α-synuclein (middle) and ∆N-α-synuclein (bottom). Black lines represent least-square fits to the data. The residuals of the fits are shown below each set of bio-layer interferometry curves. Each individual kinetic experiment was run twice in triplicates with similar results. Source data

Extended Data Fig. 5 Interaction between α-synuclein and cellular extracts.

a, Overlay of two-dimensional [15N, 1H]-NMR spectra of 50 µM [U-15N]-α-synuclein in the absence (black) and presence (green) of 25 mg ml−1 of E. coli cell extract. b, Overlay of two-dimensional [15N, 1H]-NMR spectra of 50 µM [U-15N]-α-synuclein in the absence (black) and presence of 50 mg ml−1 mammalian MDCK-II cell extract (blue-green). c, Overlay of two-dimensional [15N, 1H]-NMR spectra of 50 µM [U-15N]-α-synuclein in the absence (black) and presence (green) of 50 mg ml−1 mammalian HEK293 cell extract. d, Residue-resolved combined chemical-shift perturbations of the α-synuclein amide moieties in E. coli cell extract (green), mammalian MDCK-II cell extract (blue) and mammalian HEK293 cell extract (green), all relative to aqueous buffer. Broken lines indicate a significance level of two s.d. from the mean. Experiments in ac were done in duplicates with similar results. Source data

Extended Data Fig. 6 LUVs and the chaperone SecB compete for α-synuclein binding.

a, Residue-resolved backbone amide NMR signal attenuation (Irel = I/I0) of α-synuclein caused by the addition of 5 mg ml−1 LUVs (125:1 molar ratio of lipid:protein; dark yellow) and after further addition of 2 equivalents of SecB (yellow). b, Residue-resolved backbone amide NMR signal attenuation (Irel = I/I0) of α-synuclein caused by the addition of 15 mg ml−1 LUVs (375:1 molar ratio lipid:protein; dark yellow) and after further addition of 2 and 6 equivalents of SecB, respectively (yellow), measured at 298 K. c, Residue-resolved backbone amide NMR signal attenuation (Irel = I/I0) of α-synuclein caused by the addition of 2 equivalents of SecB (yellow) and increasing amounts of LUVs with the following ratios: 2.5 mg ml−1, 62.5:1; 4.0 mg ml−1, 100:1; 6.25 mg ml−1, 156:1; 8.5 mg ml−1, 212.5:1. d, Schematic showing the conformational equilibrium of free α-synuclein, its chaperone-bound state and one possible conformation of its LUV-bound state (PDB 1XQ8)19. Notably, these observations are also in full agreement with related studies for HSP9012 and HSP2737. e, Dynamic light scattering (DLS) measurements of LUVs prepared from pig brain polar lipids. Two independent preparations are shown in blue and orange, respectively, with an average diameter of 110 nm. Source data

Extended Data Fig. 7 Interaction of α-synuclein and chaperones in cells.

a, Western blot analysis of the expression of α-synuclein fused to a C-terminal haemagglutinin (HA)-tag in HEK293 cells. The molecular mass marker and the band corresponding to α-synuclein–HA are indicated. With these samples, immunoprecipitation and subsequent mass-spectrometry analysis was performed (b and Fig. 2a). b, Intensity ratios of carboxy-terminally HA-tagged ∆N-α-synuclein and α-synuclein immunoprecipitation determined by relative quantitative mass-spectrometry analysis. Experiments were performed as duplicates in HEK293 cells. Identification of at least five peptides per protein was required for quantification. Data are mean. The dotted line represents an intensity ratio of 1. Proteins that belong to specific groups are highlighted in colours. The values for α-synuclein (green) as well as tubulin β4 and tubulin α1B (orange arrows from left to right) are indicated by coloured arrows. c, Efficiency of HSC70 knockdown in HEK293 cells (constitutively expressing the T-Rex repressor) stably transfected with an inducible shRNA targeting HSC70 mRNA (shHSC70). The image shows a representative semiquantitative reverse-transcription (RT)–PCR of HSC70 mRNA in cells treated with doxycycline to induce shHSC70 and geldanamycin (Gel) and radicicol (Rad) for 24 h (+). Cells transfected with a control shRNA targeting firefly luciferase (shLUC) as well as semiquantification of an unrelated chaperone (HSP40) were included as negative and loading controls. d, Semiquantification of HSC70 and HSP90 protein levels by western blot. HEK293 cells (constitutively expressing the T-Rex repressor) stably transfected with shHSC70 and shLUC were grown in normal (−) or doxycycline-containing (+) medium for HSC70 knockdown. The cells were subsequently treated with vehicle (−) or geldanamycin and radicicol for HSP90 inhibition. The constitutively expressed protein GAPDH was assayed as loading control. e, Efficiency of the combined treatment of geldanamycin and radicicol in disrupting the α-synuclein–HSP90 interaction. HEK293 cells were treated with geldanamycin and radicicol for 4 or 24 h and then electroporated with recombinant α-synuclein using the protocol for in-cell NMR experiments. Whole-cell lysates were collected and used in immunoprecipitation assays with anti-α-synuclein antibodies. The obtained precipitates were then resolved by SDS–PAGE and analysed by western blot using the indicated antibodies. In addition to HEK293 cells with normal levels of HSP90 (control cells), cells with reduced levels of HSP90 (shHSP90) were used to validate the HSP90 band. f, Inhibition of both HSP90 and HSC70 promotes aggregation of α-synuclein. The image shows a representative semiquantitative western blot of HSC70-depleted HEK293 cells treated with geldanamycin and radicicol. After 24 h of treatment, the cells were subjected to electroporation with recombinant α-synuclein and 4 h after electroporation the cells were collected and analysed by western blot. HMW and 14 kDa refer to high-molecular weight and monomeric α-synuclein species, respectively. g, h, Quantification of intracellular levels of HSP90 and electroporated α-synuclein in HEK293 cells by parallel reaction monitoring mass spectrometry. A standard curve (contained in the yellow boxes) using increasing amounts of recombinant HSP90 (g) or α-synuclein (h) enables the relative quantification of the intracellular protein levels. As surrogates for intracellular protein levels, at least four tryptic peptides of HSP90 (g) or human α-synuclein (h) were quantified. Targeted peptides are shown at the top of each plot, and at least four transitions of the y-series of the product ions were monitored over the chromatographic separation of the peptides (different colours). The determined cellular concentrations of HSP90 and α-synuclein were 30 µM and 2.5 µM, respectively (see Supplementary Methods for details of this calculation). cps, counts per second. The original and uncropped gels of a, cf can be found in Supplementary Fig. 1. Western blot and PCR experiments (a, cf) were done in duplicates, with in similar results. Source data

Extended Data Fig. 8 Sequence-specific NMR-resonance assignments of α-synuclein variants.

ac, Two-dimensional [15N, 1H]-NMR spectra of 500 µM [U-13C, 15N]-α-synuclein (grey), 450 µM [U-13C, 15N]-acetyl-α-synuclein (dark violet) and 100 µM [U-15N]-∆N-α-synuclein (dark blue). The sequence-specific resonance assignments for wild-type as well as acetylated α-synuclein obtained from three-dimensional triple resonance experiments and from chemical-shift mapping of ΔN-α-synuclein are indicated. d, e, Two-dimensional [13C, 15N]-NMR spectra of 500 µM [U-13C, 15N]-α-synuclein (grey) and 450 µM [U-13C, 15N]-acetyl-α-synuclein (dark violet). The sequence-specific resonance assignments for wild-type and acetylated α-synuclein obtained from three-dimensional triple resonance experiments are indicated. f, Residue-resolved combined chemical-shift perturbations of the amide moieties for acetyl-α-synuclein (dark violet) and ΔN-α-synuclein (dark blue) versus wild-type α-synuclein. g, Residue-resolved combined chemical-shift difference of the carbonyl-amide moieties for acetyl-α-synuclein (dark violet) versus wild-type α-synuclein. [15N, 1H]-NMR spectra in ac were measured five times and [13C, 15N]-NMR spectra (d, e) were measured in duplicates, all yielding similar results. Source data

Extended Data Fig. 9 Sequence-specific NMR-resonance assignments of methionine-oxidized and tyrosine-phosphorylated α-synuclein variants.

ac, Two-dimensional [15N, 1H]-NMR spectra of 100 µM oxidized [U-15N]-α-synuclein (light grey), 100 µM oxidized [U-15N]-acetyl-α-synuclein (violet) and 100 µM oxidized [U-15N]-ΔN-α-synuclein (blue). The sequence-specific resonance assignments from chemical-shift mapping and published assignments of the oxidized state23 are indicated. Oxidized methionines are highlighted in red. d, Residue-resolved combined chemical-shift differences of the amide moieties for oxidized α-synuclein (light grey), oxidized acetyl-α-synuclein (violet) and oxidized-ΔN-α-synuclein (blue) relative to their respective reduced states. Colours as in ac. Arrows indicate the positions of the oxidized methionines. eg, Two-dimensional [15N, 1H]-NMR spectra of 50 µM [U-15N]-mono-phospho-α-synuclein (red-brown), 50 µM [U-15N]-tri-phospho-α-synuclein (brown) and 50 µM [U-15N]-tetra-phospho-α-synuclein (dark brown). The sequence-specific resonance assignments based on published assignments for phosphorylated α-synuclein are indicated24. Phosphorylated residues are highlighted in cyan. h, Residue-resolved combined chemical-shift differences of the amide moieties for the phosphorylated α-synuclein variants relative to wild-type α-synuclein. Colours as in eg. Arrows indicate the positions of the phosphorylated tyrosines. [15N, 1H]-NMR spectra of the different modified α-synuclein variants were measured several times (n = 4) yielding similar results. Source data

Extended Data Fig. 10 Mechanism of chaperone-controlled regulation of α-synuclein function, conformation and localization in mammalian cells.

Cellular chaperones (yellow) interact with the N-terminal segment of α-synuclein (red), thus actively regulating its functional species by shifting conformational equilibria. Impairment of the natural α-synuclein–chaperone ratio or abrogation of the α-synuclein–chaperone interaction by post-translational modifications can lead to the formation of pathological species, including the accumulation of α-synuclein at mitochondria.

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Uncropped gels of the experiments shown in Extended Data Figure 7.

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Burmann, B.M., Gerez, J.A., Matečko-Burmann, I. et al. Regulation of α-synuclein by chaperones in mammalian cells. Nature 577, 127–132 (2020). https://doi.org/10.1038/s41586-019-1808-9

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