α-Synuclein regulation by chaperones in mammalian cells

α-Synuclein–chaperone interaction at atomic detail

Based on previous findings that molecular chaperones share common patterns of client recognition^10,11, we characterized the interactions of an array of molecular chaperones with α-Synuclein. The array included human Hsc70 and Hsp90β, and bacterial chaperones SecB, Skp, SurA, and Trigger Factor, featuring strongly diverse architectures¹⁰. Any of these chaperones interferes functionally with α-Synuclein aggregation in a Thioflavin T (ThT) assay^6,8,12, already at 1:20 sub-stoichiometry, and with stronger effects at 1:10 ratios (Figs. 1a–c). The known Hsp90β inhibitors Geldanamycin and Radicicol (referred to onwards as drugs) decreased the chaperoning effect of Hsp90β (Fig. 1c), in line with the known mechanism of these drugs^13,14. We determined the segments of α-Synuclein interacting with the individual chaperones at the atomic level by measuring NMR signal intensity attenuations and chemical shift perturbations in 2D [¹⁵N,¹H]-NMR spectroscopy. For all six chaperones, the effects were most pronounced for twelve amino acid residues at the N-terminus and for six residues around Tyr39, indicating a direct albeit transient intermolecular interaction via these two segments, which are thus identified as the canonical chaperone-interaction motif of α-Synuclein (Fig. 1d–g; Extended Data Figs. 1,2). Inhibiting Hsp90β by drugs affected the interaction with α-Synuclein partially and for Hsc70 the interaction was observed in the ADP– and the ATP–bound, but not in the apo–state (Fig. 1g; Extended Data Fig. 3), in line with earlier reports^6,15,6,16 (see Supplemental discussion). Importantly, for all six chaperones, the interaction is observed at protein concentrations of 100 μM, far away from possible non-specific effects of macromolecular crowding. We probed such non-specific effects with high concentrations of either bovine serum albumin (BSA) or ubiquitin. No signal attenuations were observed for 150–310 mg/ml ubiquitin, ruling out macromolecular crowding effects. For high concentrations of BSA the canonical chaperone interaction signature is observed (Fig. 1g; Extended Data Figs. 3d–j), due to BSA’s weak molecular chaperone function¹⁷. Together, the experiments on an array of six chaperones and two control proteins revealed a canonical chaperone interaction for α-Synuclein at the amino-terminus and around Tyr39, transient in nature. Notably, it comprises the two locally most hydrophobic segments of α-Synuclein (Extended Data Fig. 3k,l), indicating an importance of hydrophobic residues for the chaperone interaction.

Towards characterizing the physiological role of chaperone–α-Synuclein interactions, we determined the affinity of α-Synuclein to Hsc70_ADP, SecB, and Skp by Bio-Layer Interferometry (BLI). α-Synuclein binds each of these chaperones with affinities ranging 1–2 μM (Extended Data Fig. 4 and Supplementary Table S1). The variant ∆N–α-Synuclein lacking 10 amino-terminal residues, has an affinity decreased by two orders of magnitude, validating this segment as part of the interaction site. At the reported cellular concentrations of α-Synuclein in neuronal synapses of ~50 μM with a combined concentration of Hsp70/Hsp90 chaperones of ~70μM¹⁸, about 90% of cellular α-Synuclein can thus be chaperone-bound.

We then analyzed published data on the NMR intensity profile of α-Synuclein inside living mammalian cells, which strikingly feature the canonical chaperone interaction signature⁹. Because this pattern was suggested to arise from interactions with cellular membranes, we probed the interaction of α-Synuclein with soluble cellular extracts. α-Synuclein in either 25 mg/ml E. coli cell-extract or 50 mg/ml soluble extracts from two mammalian cell lines, HEK-293 or MDCK-II, showed the canonical chaperone interaction pattern (Fig. 1h; Extended Data Figs. 5a–d), thus experimentally reproducing the in-cell interaction pattern in the absence of membranes⁹. Furthermore, we characterized the interaction pattern of α-Synuclein with lipid bilayer membranes in vitro¹⁹. Titrating LUVs (Large unilamellar vesicles) to α-Synuclein in a 125:1 lipid:protein ratio lead to a uniform NMR signal decrease for residues 1–90 (Extended Data Fig. 6a), in full agreement with published reports^9,19. Adding 2–6 equivalents of SecB to α-Synuclein–LUV solutions restored the chaperone signature, whereas the reverse experiment, addition of LUVs to an existing SecB–α-Synuclein complex led to global signal attenuation for residues 1–90 with substantially reduced effect, indicating that LUVs and SecB mutually compete for α-Synuclein binding (Extended Data Fig. 6). Overall, α-Synuclein thus populates an equilibrium between its free state, its membrane-bound state, and its chaperone-bound state, with the latter two being mutually exclusive. The emerging hypothesis that in mammalian cells α-Synuclein is dominantly in contact with chaperones rather than with lipid bilayers was supported by an experimental determination of the interactome of the α-Synuclein amino-terminus in mammalian cells by chemical cross-linking and mass-spectrometry. The interactome consists of a large number of molecular chaperones in the range of 30–75% abundance, including several Hsp90-variants and six Hsp70-variants (Fig. 2a; see Supplemental for details).

In-cell NMR spectroscopy

Next, we carried out in-cell NMR experiments to study the interaction of α-Synuclein with chaperones inside living mammalian cells at atomic resolution. [U-¹⁵N]–α-Synuclein was delivered into HEK-293 cells at a concentration of 3–10 μM, yielding intensity patterns characteristic for mammalian cell-lines⁹ (Figs. 2b–c), comprising the canonical chaperone–interaction signature and its transient nature. Multiple molecular chaperones are present in the cell, with mutually overlapping functions and “clientomes”²⁰. In line with our in vitro chaperone array, we probed the two constitutively most expressed eukaryotic chaperones Hsc70 and Hsp90β. When [U-¹⁵N]–α-Synuclein was delivered into cells with reduced Hsc70 levels (Extended Data Figs. 7c,d), the NMR intensity profile resembled the one observed for normal cells, suggesting a functional redundancy with other cellular chaperones (Figs. 2d,e). Next, we treated cells with the Hsp90-inhibiting drugs, and found that the canonical chaperone interaction motif showed increased intensities compared to untreated cells (Fig. 2d). This suggests that Hsp90 chaperones physically interact with α-Synuclein in normal cells transiently, and that this interaction is lost upon drug treatment. Immunoprecipitation assays confirmed an almost complete loss of this interaction at 24 hours post-treatment (Extended Data Fig. 7e). Finally, we inhibited both Hsc70 and Hsp90β chaperones simultaneously, observing a moderate effect on the canonical interaction motif already at 4 hours post-treatment, when a significant fraction of Hsp90 still remains bound to α-Synuclein (Extended Data Fig. 7e). At this time point, a low but measurable amount of free intracellular α-Synuclein was observed (Fig. 2d). At 24 hours post-treatment, a dramatic global signal reduction for residues 1–90 was observed, essentially identical to the LUV interaction pattern, as well as to the profile reported for α-Synuclein binding bacterial membranes^19,21 (Figs. 2d,f). The combined inhibition of the two chaperones types Hsc70 and Hsp90 thus leads to a transient membrane interaction of α-Synuclein, absent in the cellular ground state. In parallel, upon suppressing both chaperones we observed the formation of macroscopic aggregates containing α-Synuclein (Extended Data Figs. 7f). Overall, these in-cell NMR and in vitro experiments show that in normal cells α-Synuclein is physically and transiently interacting with a pool of constitutively expressed chaperones and that this interaction dominates over α-Synuclein’s transient interaction with membranes. In typical cells including neurons^18,22, as well as in our in-cell experiments the concentration of chaperones is substantially larger than the concentrations of α-Synuclein, underlining the physiological relevance of these observations (Extended Data Figs. 7g,h).

Intracellular membrane localization

The interaction of α-Synuclein with cellular membranes upon Hsc70/Hsp90 inhibition may be a key mechanism for disease pathogenesis and we thus aimed at identifying the involved membranous organelle by co-localization. To this end, control cells and HEK-293 cells depleted of Hsc70 and treated with drugs for 24 hours, stained with mitotracker (stains mitochondria), lysotracker (stains acidic vesicles, e.g. lysosomes), or Alexa-Fluor-labeled wheat germ agglutinin (WGA, stains plasma membrane and endoplasmic reticulum), and subsequently immunostained with anti-α-Synuclein antibodies. These experiments revealed a strong co-localization of α-Synuclein with mitochondria upon depletion of chaperones (Fig. 3a–c). To further confirm this association, we carried out immunofluorescence analyses using antibodies specific for the mitochondrial marker CoxIV and α-Synuclein (Fig. 3d), as well as the mitochondrial marker mtBFP (mitochondrial blue fluorescent protein) in control and in HEK-293 cells with impaired Hsc70 and Hsp90 activity (Figs. 3e,f). Both additional approaches confirmed the localization of α-Synuclein to mitochondria upon chaperone inhibition.

Effect of posttranslational modifications

Having established the canonical chaperone interaction signature, and validated its presence in living cells, we investigated the effect of chemical modifications on the α-Synuclein–chaperone interaction. Using the chaperones Hsp90β, Hsc70_ADP, SecB, and Skp, we probed the effects of amino-terminal acetylation of α-Synuclein, the predominant form in mammalian cells^9,19. Acetylation does not interfere with the chaperone interaction (Fig. 4; Extended Data Figs. 8a–g). In contrast, ∆N–α-Synuclein features a completely reduced interaction with all chaperones, in full agreement with the BLI experiments, and revealing a synergistic effect between the amino-terminus and the stretch around Tyr39 (Figs. 4b–e). Cellular oxidative stress and reactive oxygen species imbalance are known hallmarks of Parkinson’s disease onset, leading to oxidative modification of α-Synuclein². Titrating of Hsp90β, Hsc70_ADP, SecB or Skp to methionine-oxidized α-Synuclein²³ showed that oxidation of Met1 and Met5 abolish the amino-terminal interaction (Fig. 4, Extended Data Fig. 9). To explore the effects of phosphorylation on chaperone interaction, we employed in vitro tyrosine-phosphorylation with different kinases^5,24 (Fig. 4; Extended Data Fig. 9). Titration of SecB, Skp, Hsp90β, or Hsc70_ADP to either tetra-phosphorylated or Tyr39-mono-phosphorylated α-Synuclein resulted in elimination of the chaperone interaction, whereas Tyr125-Tyr133-Tyr136-tri-phosphorylated α-Synuclein showed the chaperone interaction pattern of unmodified α-Synuclein (Fig. 4). Tyr39 phosphorylation thus has a specific inhibitory effect towards chaperone interaction, providing a direct rationale for in vivo studies showing that up-regulation of c-Abl correlates strongly with Tyr39-phosphorylation and disease progression in Parkinson’s disease^5,25.

Conclusion

Overall, in this work we have identified a functional mechanism for regulation of α-Synuclein by chaperones in mammalian cells through transient binding (Extended Data Fig. 10). Molecular chaperones bind the IDP α-Synuclein via a canonical motif, based on recognizing intrinsic biophysical features. The interaction vanishes upon inhibition of two main chaperones, resulting in transient membrane interactions and an accumulation of α-Synuclein at mitochondria, a major component of Lewy bodies^26,27. A functional model emerges, where transient chaperone-interacting forms are the dominant species of α-Synuclein in healthy cells, making chaperones a master regulator of the cellular states of α-Synuclein. It predicts that changes in the cellular levels of either chaperones or α-Synuclein, or the modulation of their interaction will disturb the homeostatic balance, eventually causing Parkinson’s disease. Notably, this model agrees with a plethora of reported experimental observations (see Supplemental for an extended discussion), including that the ratio of α-Synuclein to chaperone is impaired by familial Parkinsonism and that oxidative stress can lead to an increase of α-Synuclein Tyr39-phosphorylation^5,25, which interferes with chaperone binding. The model shows further how modulation of chaperone activity might prevent the formation of oligomeric α-Synuclein, leading to mitochondrial membrane disruption²⁸ and also accounts for recent reports that impairment of mitochondria may constitute an important factor in Parkinson’s disease^29–31.

Extended Data

Supplementary Material