
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01882-0
10.1016/j.isci.2024.110657
110657
Article
HSPB6: A lipid-dependent molecular chaperone inhibits α-synuclein aggregation
Secco Valentina 13
Tiago Tatiana 13
Staats Roxine 2
Preet Swapan 2
Chia Sean 2
Vendruscolo Michele mv245@cam.ac.uk
2∗
Carra Serena serena.carra@unimore.it
14∗∗
1 Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
2 Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK
∗ Corresponding author mv245@cam.ac.uk
∗∗ Corresponding author serena.carra@unimore.it
3 These authors contributed equally

4 Lead contact

03 8 2024
20 9 2024
03 8 2024
27 9 1106573 5 2024
9 6 2024
31 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

The process of protein misfolding and aggregation is associated with various cytotoxic effects. Understanding how this phenomenon is regulated by the protein homeostasis system, however, is difficult, since it takes place through a complex non-linear network of coupled microscopic steps, including primary nucleation, fibril elongation, and secondary nucleation, which depend on environmental factors. To address this problem, we studied how the aggregation of α-synuclein, a protein associated with Parkinson’s disease, is modulated by molecular chaperones and lipid membranes. We focused on small heat shock proteins (sHSPs/HSPBs), which interact with proteins and lipids and are upregulated during aging, a major risk factor for protein misfolding diseases. HSPBs act on different microscopic steps to prevent α-synuclein aggregation, with HSPB6 showing a lipid-dependent chaperone activity. Our findings provide an example of how HSPBs diversified their mechanisms of action to reach an efficient regulation of protein misfolding and aggregation within the complex cellular environment.

Graphical abstract

Highlights

• α-synuclein aggregation can be modulated by lipids and pH

• HSPBs evolved to inhibit α-synuclein aggregation through multiple mechanisms

• HSPB6 shows a lipid-dependent chaperone activity

• HSPB3, 5, 7, and 8 preferentially act on fibril elongation and secondary nucleation

Chemistry; Biological sciences; Cell biology

Subject areas

Chemistry
Biological sciences
Cell biology
Published: August 3, 2024
==== Body
pmcIntroduction

α-synuclein (α-syn) is an intrinsically disordered protein with an N-terminal domain (residues 1–60) that adopts an α-helical conformation upon binding to phospholipid membranes, a central hydrophobic non-β-amyloid component (NAC) domain (residues 61–95) that adopts β sheet structures upon aggregation, and a negatively charged C-terminal domain (residues 96–140) that can bind to calcium ions.1 α-syn is highly expressed in the brain, where it is associated with synaptic vesicles and regulates synaptic transmission.2,3 α-syn preferentially binds to lipid membranes and regulates different aspects of vesicular dynamics, from vesicle tethering to fusion and exocytosis.4

Like other intrinsically disordered peripheral membrane proteins, α-syn exists in a soluble and membrane-bound form. Unbound α-syn and monomeric α-syn adopt partially folded intermediate conformations; upon binding to lipid membranes, α-syn adopts an α-helical structure.5 However, binding to lipids represents a double-edged sword, because it can promote α-syn oligomerization and amyloid formation, which contribute to the development of a group of neurodegenerative disorders called synucleinopathies.6,7,8 Synucleinopathies include Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple-system atrophy (MSA). PD is characterized by intracellular deposits called Lewy bodies (LBs),9 which are heterogenous in composition and not only contain amyloid fibrils of α-syn but also other proteins such as molecular chaperones and large amounts of lipids and membranous organelles.10 Of note, several disease-linked mutations are found in the α-syn N-terminal membrane-binding region and affect α-syn association with lipid droplets and vesicles, promoting its aggregation.11 Moreover, lipid alterations have been identified in the brain and plasma of patients affected by PD, and mutations in the glucosylceramidase-beta (GBA) gene, which affect the sphingolipid metabolism, are an important risk factor in PD and DLB.12 Additional genes involved in glycerophospholipid and sphingolipid metabolism, namely the PLA2G6 and SCARB2 genes, are also considered risk factors for PD.13 Taken together, these findings support a lipid-centric view of α-syn aggregation and suggest that identifying approaches that limit α-syn lipid-induced aggregation may open new therapeutic avenues.14

α-syn aggregation is a multi-step process that involves several interconvertible conformational states, transient oligomeric states, and prefibrillar species, which are influenced by environmental factors, such as lipids and pH.15 The main steps that can be identified for α-syn aggregation: (1) primary nucleation, in which monomers assemble to form disordered oligomeric species; oligomer conversion, in which these oligomeric species convert into ordered fibrillar seeds rich in β structures; (2) elongation, in which the seeds grow to generate protofibrils and fibrils; and (3) secondary nucleation, a step in which monomers assemble on the surface of fibrils to generate new fibrils and new oligomeric species.15 An additional pathway that can promote α-syn aggregation has been recently identified: liquid-liquid phase separation of α-syn into condensates that can mature into amyloid hydrogels containing α-syn oligomers and fibrillar species.16,17,18 Of note, because of the high concentration within condensates, α-syn primary nucleation can be readily observable at physiological pH, which is followed by the rapid growth and amplification of α-syn aggregates.19

Molecular chaperones, mainly heat shock proteins (HSPs), combat protein aggregation, delaying the growth of amyloid fibrils20 and preventing the conversion of liquid-like condensates into amyloid hydrogels.21 For example, the ATP-dependent chaperone HSP70, a master regulator of protein homeostasis (proteostasis), interacts with α-syn monomers, oligomers, and prefibrillar species, protecting against α-syn-mediated toxicity in cellular and animal models of PD.22 In addition, a molecular chaperone complex containing Hsc70 and the co-chaperone Hsp40/DnaJB1 and Apg2 can remove α-syn monomers from the fibril ends, reverting the aggregation process.23 Also the small HSPs/HSPBs, which are ATP-independent chaperones and include 10 members in humans (HSPB1-HSPB10),24 can prevent α-syn aggregation. For example, HSPB1 (Hsp27), HSPB2 (MKBP), HSPB3 (HSPL27), HSPB5 (αB-crystallin), and HSPB8 (Hsp22) bind to α-syn fibrils and inhibit fibril elongation and maturation.25,26,27,28,29,30 In addition, HSPBs colocalize with LBs, and their expression levels are upregulated in brain regions characterized by neurodegeneration in protein misfolding diseases such as PD.31,32 Together these observations support the view that molecular chaperones, including HSPBs, play an active role in preventing α-syn aggregation. Yet these studies have focused on a protein-centric view of α-syn aggregation and the mode of action of molecular chaperones. However, molecular chaperones can bind to lipids,33,34,35 and α-syn association with lipids enhances the rate of aggregation.7,36 We still poorly understand the mechanisms regulating the binding of molecular chaperones to lipids and their functional roles. The association of HSPs with lipids has been suggested to stabilize membranes upon stress conditions, including heat shock, to regulate endo-lysosomal trafficking, endocytosis, and cancer cell invasion, with important physiological and pathological implications.35,37,38,39 A number of proteins, including the fatty acid-binding proteins (FABPs), have been proposed to act as “lipid chaperones”, a term that refers to molecular chaperones that have lipids, rather than proteins, as substrates.40 In addition, the “lipid-chaperone hypothesis” states that free lipid molecules can interact with proteins forming water-soluble lipid-protein complexes that promote protein anchoring to the membranes, influencing their stability.41 Yet, whether or not the anti-aggregation activity of molecular chaperones depends on specific types of lipid membranes remains unknown. Here, we addressed this question focusing on HSPBs and α-syn as a model protein.

HSPBs are characterized by a low molecular weight (12–43 kDa) and the presence of a highly conserved α-crystallin domain of 80–100 amino acids, flanked by less conserved N-terminal and C-terminal intrinsically disordered domains.42 All these regions are involved in regulating the assembly of HSPB monomers into polydisperse oligomers that are thought to act as a cellular chaperone reservoir.43 Human HSPBs show a high structural diversity and can assemble into dynamic oligomers of different size and compositions. HSPB1, HSPB4, and HSPB5 co-assemble into polydisperse oligomers, while HSPB6, HSPB7, and HSPB8 mainly exist in form of dimers44,45 and HSPB3 can form tetramers with HSPB2 at a 1:3 ratio.46 Upon stress conditions, such as changes in the temperature, pH, and ion concentrations and in presence of unfolded and misfolded proteins, HSPB assemblies dissociate into smaller oligomers, dimers, and monomers giving rise to an HSPB ensemble with chaperone activity that can bind to and neutralize non-native and aggregation-prone intermediates, preventing their irreversible aggregation.42 Recent studies that analyzed the accessible surface areas of small HSPs of different origins showed that upon dissociation of the larger oligomers, the α-crystallin domain of the resultant monomers and dimers are partially unfolded and display enhanced chaperone activity.47 Thus, the dynamic release of disordered monomers/dimers has been proposed to represent a common mechanism of action for small HSPs.47 Nevertheless, due to the ability of HSPBs to interact with each other to form homo- and hetero-oligomers of variable size, which influences their affinity for different substrates, one would not expect their chaperone activity to be described with one unifying mechanism. Rather, distinct mechanisms may exist dependent on the type of HSPB, the type of substrate and the surrounding environment, such as pH, salinity and presence of lipids.48

Using a three-pronged kinetic approach previously developed,7,11 we tested here the ability of selected HSPBs to inhibit α-syn lipid-induced aggregation, as well as fibril growth in absence of lipids. The chaperone activity of HSPB1, HSPB4, and HSPB5 on α-synuclein has been characterized in detail by previous groups.25,26,27,28,29,49,50,51,52,53,54 Here, we focused on the less-well studied HSPB members that tend to mainly form dimers/small oligomers. Since both HSPB1 and HSPB5 typically form large oligomers and were both previously shown to bind to lipids,35 we selected HSPB5 for comparative analysis, while focusing on the other less characterized members of the family that mainly form dimers/smaller oligomers. Our findings identify HSPB6 as a lipid-dependent molecular chaperone, since it could efficiently prevent α-syn aggregation only in presence of lipid membranes. Instead, the other HSPBs tested showed stronger chaperone activity in presence of monomeric α-syn and preformed α-syn fibrils, and a moderate chaperone activity in presence of lipid membranes. These results highlight the diversity and complexity of HSPBs, which diversified their mechanisms of action to reach an efficient regulation of protein aggregation within the complex lipid-enriched cellular environment. We anticipate that the classification of poor and efficient chaperones based on protein-only assays will warrant further investigation, and the identification of other lipid-dependent molecular chaperones similar to HSPB6 may represent the first step toward new potential therapeutic avenues.

Results

HSPBs inhibit α-syn lipid-induced aggregation

A three-pronged approach is proposed to dissect the key steps of α-syn aggregation, namely lipid-induced aggregation, elongation, and secondary nucleation (Figure 1). The assay to monitor α-syn aggregation induced by lipids has been developed to study α-syn aggregation at physiological concentrations, which are estimated to be lower than 30–60 μM and in presence of small unilamellar vesicles (SUVs), which trigger α-syn amyloid fibril formation (Figure 1, left panel).7,11,55 Primary nucleation is followed by fibril growth by elongation, which can be studied using a second assay (in absence of lipids) through addition of monomeric α-syn to the growing extremity of existing fibrils at high concentration (Figure 1, middle panel, high seed). Finally, secondary nucleation events that occur via formation of surface-catalyzed aggregates and lead to fibril amplification are monitored under conditions of mildly acidic pH and low fibril concentration (Figure 1, right panel, low seed).11,56 These mildly acidic environmental conditions mimic the low pH (5.5) that characterizes dopaminergic neurons, which express highest levels of α-syn.57 Using this three-pronged approach we evaluated the impact of individual HSPBs on α-syn aggregation, identifying the contribution of specific environmental factors that are biologically relevant such as lipid composition and pH variation.Figure 1 Schematics of the three-pronged approach used to characterize α-syn aggregation

Three different assays were used to probe: (left) lipid-induced aggregation, where monomeric α-syn forms growth-competent nuclei due to the interaction with lipid surfaces; (middle) elongation, where monomeric α-syn interacts with existing preformed fibrils at high concentrations (high seed) and pH 6.5, forming longer fibrils; (right) secondary nucleation, where monomeric α-syn interacts with existing preformed fibrils at low concentrations (low seed) and pH 5.5. Secondary nucleation refers to a surface catalyzed mechanism that leads to the formation of toxic oligomeric species in the presence of amyloid fibrils. This process is accelerated at mildly acidic pH, generating new fibrils and new oligomeric species.6,7,11

In these assays, α-syn amyloid fibril formation is followed over time by measuring the increase of thioflavin T (ThT) fluorescence. We previously reported that the kinetics of α-syn aggregation are accelerated upon incubation of α-syn monomers with SUVs prepared from the model membrane lipid DMPS (1,2-dimyristoyl-sn-glycero-3-phospho-L-serin)6,7,58 (Figure 1, left panel; Figure 2, black curves). Although of limited physiological relevance, DMPS SUVs represent a well-established model to study α-syn lipid-induced aggregation and were used as the reference system here. The diameter of DPMS lipid vesicles was measured prior to their use for each experiment; a representative measure of DMPS lipid in shown in Figure S1A. No increase in the ThT fluorescence signal was detected in absence of DMPS under the experimental conditions used, while α-syn amyloid fibril formation started already after 5 h of co-incubation with DMPS SUVs and reached a plateau after circa 25 h (Figure 2A, black curve), when the molecules of monomeric α-syn, initially present in the reaction mixture, were incorporated into the growing fibril. Thus, the lower the time required to reach the plateau and the steeper the curve slope, the higher the rate of α-syn aggregation. We then co-incubated DMPS with α-syn and either HSPB3, HSPB5, HSPB6, HSPB7, or HSPB8 starting with an HSPB:α-syn ratio of 1:100. HSPB6 completely inhibited α-syn lipid-induced aggregation on the timescale that we monitored (100 h) (Figure 2A). Instead, HSPB3, HSPB5, HSPB7, and HSPB8 delayed α-syn lipid-induced aggregation at low stoichiometries, but with different degrees of efficacy, with HSPB3 showing the poorest chaperone activity (Figures 2A and 2B). The beginning of the aggregation occurred circa 5 h after co-incubation of DMPS with α-syn alone (Figure 2A, black curve) and was delayed to about 25–35–40 and 45 h in presence of HSPB3, HSPB7, HSPB8, and HSPB5, respectively, while it never occurred with HSPB6 (Figure 2A, colored curves). The rate at which the α-syn fibrils grow is indicated by the slope of the curve. By comparing the slope of the curves in absence or presence of HSPB3, HSPB5, HSPB7, and HSPB8, we deduced that the aggregation process proceeded with similar kinetics to the control condition (α-syn) (Figure 2A). Thus, at the HSPB:α-syn ratio of 1:100, only HSPB6 completely abrogated α-syn lipid-induced aggregation.Figure 2 HSPBs inhibit lipid-induced α-syn aggregation

(A–G) Changes in ThT fluorescence intensity when monomeric α-syn (20 μM) is incubated with 100 μM DMPS vesicles at pH 6.5°C and 30°C (DMPS:α-syn ratio is 5:1) and in the absence (black) or presence of HSPBs (colored lines) at the following HSPB:α-syn ratios: 1:100 (0.2 μM:20 μM) (A), 1:200 (0.1 μM: 20 μM) (C), 1:400 (0.05 μM:20 μM) (E), and 1:50 (0.4 μM: 20 μM) (G). Data are presented as absolute ThT fluorescence. a.u. arbitrary units. (B, D, and F) Comparison of α-syn aggregation half-time in absence/presence of HSPBs, as per conditions detailed in A, C, and E, respectively. (A–G) Data are shown as mean ± SEM. n = 3 independent experiments. One-way ANOVA, followed by Bonferroni-Holm post-hoc test. Comparison between α-syn alone or with each HSPB is shown.

We next applied a previously proposed lipid-induced aggregation model7 to our data to compare the efficacy of the HSPBs studied. In this model, the nucleation process is assumed to occur at the surface of the vesicles and then to be followed by the growth of fibrils from lipid-bound α-syn. We calculated the half-time (t1/2) of α-syn aggregation alone or in presence of HSPBs. At the HSPB:α-syn ratio of 1:100, HSPB3 had the lowest efficacy, with an aggregation half-time of ca. 30 h (Figure 2B). HSPB6 was excluded from this analysis since it completely abrogated α-syn lipid-induced aggregation at the HSPB:α-syn ratio of 1:100 (Figure 2B). The same experiments were then conducted at higher stoichiometries using, respectively, the HSPB:α-syn ratio of 1:200 (Figures 2C and 2D) and 1:400 (Figures 2E and 2F). At the lowest HSPB:α-syn ratio of 1:50, all the HSPB tested could prevent α-syn lipid-induced aggregation (Figure 2G). HSPB efficacy was further compared by measuring the relative aggregation constant normalized for the aggregation rate, using a previously published one-step nucleation model.7 Using this model, we confirmed that HSPB3 had the lowest efficacy, with a relative aggregation rate ratio to α-syn alone of 0.10 (Figure S1B); instead, HSPB5, HSPB7, and HSPB8 reduced the lipid-induced aggregation with a relative aggregation rate ratio between 0.05 and 0.07 (Figure S1B). HSPB6 was excluded from this analysis since it completely inhibited α-syn lipid-induced primary-nucleation at the 1:100 HSPB:α-syn ratio. When using higher HSPB:α-syn ratios, we confirmed the strongest inhibitory effect of HSPB6 in presence of lipids compared to the other HSPBs tested (Figures S1C and S1D). Thus, although all HSPBs prevented α-syn lipid-induced aggregation in a dose-dependent manner, HSPB6 showed the greatest efficacy.

Considering that previous reports showed that HSPB5 and HSPB8 interact both with α-syn and lipids,26,28,33,35,54 these results are compatible with at least two possible, but not mutually exclusive, incompatible, mechanisms: HSPBs compete with α-syn for binding to lipid membranes, thus reducing α-syn lipid-induced aggregation; HSPBs bind to α-syn monomers, thus preventing their binding to the lipid surface. Alternatively, HSPBs bind to α-syn embedded in the lipids, shielding it from oligomerization and aggregation. To differentiate between these possible mechanisms, we next tested whether HSPB3, HSPB5, HSPB6, HSPB7, or HSPB8 can interact with lipid membranes.

Proteins embedding into lipid membranes affect their fluidity and melting temperature, which can be measured by monitoring changes in the polarization of the fluorescence of the probe diphenylhexatriene (DPH): the higher the anisotropy of DPH, the lower the membrane fluidity.59 We previously showed that DMPS SUVs convert at 41°C from a gel phase bilayer with solid hydrocarbon chain into a liquid crystalline bilayer with fluid hydrocarbon chain.6 In presence of α-syn, the melting temperature of DMPS SUVs bound to α-syn decreases and is centered at 25°C.6 Since temperature fluctuations affect lipid phase transitions, which can in turn modify the binding affinity of proteins for lipids, we measured how the microviscosity of DMPS SUVs changes in absence or presence of HSPBs by monitoring the DPH fluorescence polarization at increasing temperature (Figure S2A, brown dotted line). The impact of the various HSPBs on DHP fluorescence polarization was compared to that of α-syn, employed as a positive control, in the temperature range from 25°C to 50°C6,7 (Figure S2A, compare the brown dotted line with the black line). As expected,6 co-incubation of DPMS with α-syn decreased the DPH fluorescence polarization in the temperature range investigated (Figure S2A). HSPB3, HSPB5, and HSPB7 increased the DHP fluorescence polarization when co-incubated at temperatures ranging from circa 25°C–37°C, whereas at higher temperatures they decreased the DHP fluorescence polarization, except for HSPB7 (Figure S2A). HSPB8 and HSPB6 decreased the DPH fluorescence polarization from and above circa 30°C, with a stronger impact of HSPB6 compared to HSPB8 (Figure S2A). These data suggest that all HSPBs tested can influence the dynamical and structural properties of lipid bilayers measured via DHP fluorescence polarization at different temperatures.

We next tested whether HSPBs can influence the DPH fluorescence polarization upon co-incubation with DMPS and α-syn, starting with an HSPB:α-syn ratio of 1:1 (Figure S2B). Compared to α-syn alone (Figure S2B, black line), HSPB6 further decreased the DPH fluorescence polarization at the physiological temperature (37°C) and above; HSPB3, HSPB5, and HSPB7 increased the DPH fluorescence polarization in the temperature range from circa 37°C–50°C), while HSPB8 had no effect (Figure S2B, compare gray line with black line). Since higher DPH anisotropy is indicative of lower membrane fluidity, we conclude that, compared to DMPS SUVs incubated with α-syn alone, in the temperature range from circa 35°C–50°C, HSPB3, HSPB5, and HSPB7 tend to restore membrane fluidity, whereas HSPB6 further decreased it, showing an additive effect with α-syn, and HSPB8 had no impact. The additive effect of HSPB6, compared to DMPS and α-syn alone, was further confirmed by lowering the concentration of HSPB6 by four times (HSPB6:α-syn ratio of 1:4; Figure S2C).

In order to further elucidate how the interaction of HSPBs influences α-syn lipid-induced aggregation, we incubated DMPS SUVs with α-syn monomers and/or HSPBs at the fixed temperature of 30°C for 4 h and we measured DPH fluorescence polarization. As previously reported,6,7 co-incubation of α-syn with DMPS SUVs decreased the DPH fluorescence polarization, which corresponds to an increase of their fluidity (Figure 3A, compare black column to brown column). When incubated alone with DMPS SUVs, HSPB7 and HSPB8 slightly increased the DPH fluorescence polarization; a similar result was obtained upon co-incubation with α-syn (Figure 3A, purple and gray columns). By contrast, HSPB3 and HSPB5 slightly decreased the DPH fluorescence polarization of DMPS SUVs in absence of α-syn, while increasing it in presence of α-syn (Figure 3A, dark blue and light blue columns). Finally, HSPB6 strongly decreased the DPH fluorescence polarization of DMPS SUVs in absence and presence of α-syn (Figure 3A, green columns).Figure 3 Impact of HSPBs on the fluidity of DMPS lipid vesicles and on α-syn lipid-induced aggregation

(A) Changes in DMPS (100 μM) membrane fluidity due to presence of α-syn or HSPBs (20 μM) detected through fluorescence polarization of DPH at 30°C for 4 h (DMPS:α-syn and DMPS:HSPB ratio are 5:1). Data are shown as mean ± SEM. n = 3 independent experiments. One-way ANOVA, followed by Bonferroni-Holm post-hoc test. Comparison between DPH fluorescence polarization of DMPS alone or with α-syn and/or each HSPB studied are shown.

(B) Putative mechanisms of action of HSPBs toward α-syn lipid-induced aggregation. HSPBs could transiently associate with α-syn monomers, hence preventing their binding to lipid membranes. In addition, and not mutually exclusive, HSPBs could interact with lipid-bound α-syn, thereby shielding it from aggregation. Furthermore, HSPBs could directly interact with lipids, thus reducing the association of α-syn with lipid membranes. The latter seems to be the preferred mechanism of action of HSPB6.

These data confirm the importance of lipids in the mechanism of action of HSPB6 and support the idea that HSPBs reduce α-syn lipid-induced aggregation with distinct mechanisms of action. Plausible mechanisms of action that may explain our results include: (1) HSPB binding to free α-syn monomers, thereby inhibiting their embedding into the lipids; (2) HSPB binding to lipid-bound α-syn, thereby shielding it from oligomerization and further aggregation (Figure 3B). Concerning HSPB6, it has been shown that transient complex between α-syn monomers and free lipid molecules, which are in equilibrium with the lipid vesicles, may form. These lipid-α-syn complexes are water-soluble and have increased hydrophobicity; this, in turn, enhances their insertion in the lipid membranes, promoting the growth of amyloid fibrils on the lipid membrane surfaces.41 By binding to either free lipids or lipid membranes, HSPB6 may prevent the formation of these transient water-soluble lipid-α-syn complexes, decreasing the binding affinity of α-syn for lipids and delaying the fibril growth.

Phosphorylation of serine HSPB6 enhances its inhibitory effect on α-syn lipid-induced aggregation

HSPB6 is the only member of the HSPB family that contains an RRXS consensus sequence recognized by the protein kinases PKA and PKG.60 The HSPB6 RRXS motif contains serine residue 16 (S16), which can be phosphorylated by cAMP-PKA and has been implicated in the regulation of HSPB6 interactions and functions, including smooth muscle relaxation and cardioprotection.61 pS16-HSPB6 promotes its binding to the 14-3-3 protein, competing with the actin depolymerizing protein cofilin.62,63 Displacement of cofilin from 14-3-3 by pS16-HSPB6 has been suggested to modulate actin dynamics, ultimately favoring actin depolymerization and smooth muscle relaxation.63

Interestingly, 14-3-3 also interacts with α-syn to reduce its aggregation in vitro and in cells, acting at the level of preformed fibrils and aggregation intermediates.64 Recently, it has been suggested that 14-3-3 protein buffers exceeding amounts of α-syn at cholesterol rich sites, preventing lipid-induced oligomerization.65 Whether it does so alone or acting in concert with other binding partners is currently unknown. Based on these premises, here we investigated whether S16-HSPB6 can inhibit α-syn lipid induced aggregation as efficiently as non-phosphorylated HSPB6. To ascertain the impact of HSPB6 phosphorylation per se, we conducted these experiments in absence of 14-3-3. We generated non-phosphorylatable HSPB6, by replacing serine 16 with an alanine (S16A) and pseudophosphorylated HSPB6, by replacing serine 16 with aspartic acid (S16D). We also replaced serine 16 with a cysteine residue (S16C), followed by a chemical addition of the phosphate (P) group to generate a phosphorylated S16C-HSPB6 (S16C-P) (Figures S3A–S3D). We next tested their efficacy in inhibiting α-syn lipid-induced aggregation, using the ThT fluorescence assay and a starting HSPB:α-syn ratio of 1:200. While non-phosphorylatable S16A prevented the α-syn lipid-induced aggregation with an efficacy similar to that of HSPB6 wild type, pseudophosphorylated S16D and chemically phosphorylated S16C-P showed a stronger inhibitory effect (Figures 4A and 4B). Similar results were obtained increasing the HSPB:α-syn ratio to 1:400, further confirming that, in absence of 14-3-3, HSPB6 Serine 16 phosphorylation slightly enhances its inhibitory effect toward α-syn lipid-induced aggregation (Figures 4C and 4D). Dimers of phosphorylated HSPB6 interact with dimers of 14-3-3,66 with a micromolar affinity in the case of the 14-3-3 ζ isoform.67 Future studies should investigate whether co-incubation of S16-HSPB6 with different 14-3-3 isoforms influences its ability to prevent α-syn lipid-induced aggregation at cholesterol rich sites.Figure 4 S16 phosphorylation enhances the lipid chaperone activity of HSPB6

(A–D) Changes in ThT fluorescence intensity when monomeric α-syn (20 μM) is incubated with 100 μM DMPS vesicles at pH 6.5°C and 30°C and in the absence (black) or presence of HSPB6 wild-type, non-phosphorylatable HSPB6 (S16A), pseudophosphorylated HSPB6 (S16D) and chemically phosphorylated HSPB6 (S16C-P) at the HSPB:α-syn ratios 1:200 (0.1 μM:20 μM) (A) and 1:400 (0.05 μM:20 μM) (C). (B and D) Comparison of α-syn aggregation half-time, in the presence of HSPB6, S16A, S16D, or S16C-P as per conditions in detailed in A and C, respectively. (A–D) Data are shown as mean ± SEM. n = 3 independent experiments. One-way ANOVA, followed by Bonferroni-Holm post-hoc test. Comparison between HSPB6 wild type and either S16A, S16D, or S16C-P is shown.

HSPB3, HSPB5, and HSPB7 delay α-syn fibril elongation

Upon assembly, α-syn fibrils grow by elongation, which can be measured using ThT fluorescence by incubating at neutral pH preformed fibrils with free α-syn monomers in absence of SUVs (Figure 1, middle panel).6,7,11 With this elongation assay, we tested the efficacy of HSPBs to prevent α-syn fibril growth, using different HSPBs:α-syn ratios. The results obtained with this assay, in absence of lipids, will be instrumental for the interpretation of the ones obtained using the α-syn lipid-induced aggregation assay. Importantly, in this fibril elongation assay we used 1 μM of α-syn seeded fibrils, 20 μM of α-syn monomers, and progressively decreasing concentrations of HSPBs, thus enabling us to extrapolate the affinity of HSPBs toward the preformed fibril extremities versus the free α-syn monomers (in large excess compared to the preformed fibrils). At the 1:1, 1:2, and 1:4 HSPBs:α-syn ratios, all HSPBs tested delayed the elongation of α-syn preformed fibrils, although with different kinetics (Figures 5A–5C). To enable direct comparison of the efficacy of the HSPBs, the measured elongation rates were normalized relative to the one of α-syn alone (Figure 5D). HSPB3, HSPB5, and HSPB7 inhibited α-syn elongation with a relative fibril elongation ratio of approximately 0.1, independently of the HSPBs:α-syn ratio. HSPB8 delayed fibril elongation with a relative fibril elongation ratio of 0.2–0.6, based on HSPB8 concentration: the lower HSPB8 concentration (1:4 ratio), the lower the efficacy (Figure 5D). Concerning HSPB6, its efficacy in inhibiting α-syn fibril elongation was poorer compared to the one of HSPB8 (Figure 5D); of note, when HSPB6 concentration was 4-times lower than the one of monomeric α-syn, HSPB6 had almost no effect (Figure 5D, 1:4 ratio).Figure 5 HSPBs delay α-syn fibril elongation

(A and B) Changes in ThT fluorescence intensity when monomeric α-syn (20 μM) is incubated with 1 μM preformed fibrils at pH 6.5°C and 37°C and in the absence (black) or presence of HSPBs at three different HSPB:α-syn ratios: 1:1 (20 μM: 20 μM) (A), 1:2 (10 μM:20 μM) (B), and 1:4 (5 μM:20 μM).

(C) Data are reported as absolute ThT fluorescence. a.u. arbitrary units.

(D) Relative rate of α-syn fibril elongation, determined as previously described.11

(E–I) Changes in ThT fluorescence intensity as described in A, but using the following HSPB:α-syn ratios: 1:200 (0.1 μM: 20 μM), 1:100 (0.2 μM: 20 μM), 1:50 (0.4 μM: 20 μM), and 1:20 (1 μM: 20 μM). (A–I) Data are shown as mean ± SEM. n = 3 independent experiments.

(J) Putative mechanism of action of HSPBs on fibril elongation.

Of note, the lipid-induced aggregation assay was performed using higher HSPBs:α-syn ratios, ranging from 1:100 and 1:400 (Figure 2). We then repeated the experiments lowering further the concentration of HSPBs and using 1:20, 1:50, 1:100, and 1:200 HSPBs:α-syn ratios (Figures 5E–5I), enabling the comparison between the efficacy of HSPBs in preventing α-syn aggregation in absence and presence of lipids. HSPB3, HSPB5, and HSPB7 could still efficiently delay α-syn fibril elongation even at the highest 1:200 ratio, while HSPB6 was completely inactive at the higher ratios and showed only poor activity at the 1:20 ratio (Figures 5E–5H). By contrast, at the 1:200 ratio, HSPB6 was by far more efficient in preventing monomeric α-syn lipid-induced aggregation. Concerning HSPB8, its efficacy was concentration-dependent and progressively decreased with increasing amounts of free α-syn monomers, being lost at the highest ratio (Figure 5I). Based on these data, we conclude that HSPB3, HSPB5, and HSPB7 efficiently prevent fibril elongation, in a wide concentration. This observation suggests that they would bind with higher affinity to the fibril surface and extremities, rather than to free α-syn monomers (Figure 5J). Of note, previous work identified higher binding affinity of HSPB5 for the length of the fibril and lower affinity for the fibril ends.54,68 Instead, HSPB8, whose efficacy is dose dependent and decreases with increasing concentrations of free α-syn monomers, would preferentially bind to the latter (Figure 5J). Finally, the comparison of the results obtained at the higher ratios 1:100 and 1:200 in absence and presence of lipids supports the idea that HSPB6 interacts weakly with monomeric α-syn (Figure 5J), and acts instead as a lipid-dependent molecular chaperone.

HSPB3 and HSPB7 inhibit the rate of α-syn fibril amplification under mildly acidic pH

We next tested the ability of HSPBs to inhibit the formation of α-syn higher order assemblies, which have been observed to occur via secondary nucleation upon incubation of α-syn fibrils and free monomers at mildly acidic pH11,69 (Figure 1, right panel). Since the secondary nucleation assay is performed at pH 5.5, we studied the folding and stability of the various HSPBs at acidic pH using circular dichroism (CD). Previous work showed that HSPB5 is sensitive to pH changes. Lowering the pH from 7.5 to 6.5 promotes dimer to monomer dissociation and the formation of polydisperse oligomers with increased holdase capacity.70 However, lowering the pH from 6.5 to 5.5 affected the CD spectra of HSPB5, suggesting protein destabilization, which may in turn affect its chaperone function (Figure S4B). Due to the high sensitivity to lower pH and to avoid data misinterpretation, we did not assess HSPB5 efficacy in inhibiting secondary nucleation at mildly acidic pH. Instead, the CD spectra of HSPB3, HSPB6, HSPB7, and HSPB8 were similar regardless of the acidic pH (Figures S4A–S4E), enabling us to study their efficacy in inhibiting α-syn secondary nucleation at pH 5.5. The secondary nucleation assay consists in the acquisition of ThT fluorescence upon co-incubation at pH 5.5 of 50 nM α-syn seeded fibrils, 20 μM α-syn monomers, and progressively decreasing concentrations of HSPBs. The use of a large excess of α-syn monomers compared to the preformed fibrils enables to gain insights into HSPB affinity on the different states of α-syn. To quantify differences in the efficacy of the HSPBs, we performed the measures using three HSPBs:α-syn ratios: 1:1, 1:2, and 1:4. HSPB3 and HSPB7 strongly inhibited α-syn secondary nucleation regardless of the ratio used (Figures 6A–6C). HSPB8 showed a moderate and dose-dependent effect, while HSPB6 showed the lowest efficacy compared to the other members of the family tested (Figures 6A–6C). Similar to what we observed using the elongation assay (Figure 5I), HSPB8 chaperone activity inversely correlated with the concentration of free α-syn monomers (Figure 6D), suggesting that HSPB8 inhibits α-syn aggregation by preferentially binding to and neutralizing its monomeric forms. By contrast, HSPB3 and HSPB7, whose efficacy is not influenced by the presence of exceeding monomeric free α-syn, would preferentially bind to α-syn fibrils (Figure 6D), in agreement with the observations from the elongation assay.Figure 6 HSPBs delay α-syn secondary nucleation

(A–C) Changes in ThT fluorescence intensity when monomeric α-syn (20 μM) is incubated with 50 nM preformed fibrils at pH 5.5°C and 37°C and in the absence (black) or presence of HSPBs at three different HSPB:α-syn ratios: 1:1 (20 μM: 20 μM) (A), 1:2 (10 μM:20 μM) (B), and 1:4 (5 μM:20 μM) (C). Data are shown as absolute ThT fluorescence. a.u. arbitrary units. Data are shown as mean ± SEM. n = 3 independent experiments. (D) Putative mechanism of action of HSPBs on secondary nucleation.

The inhibitory effect of HSPB6 on α-syn lipid-induced aggregation correlates with its affinity for lipids

The integrated analysis of the results obtained in absence (Figures 5 and 6) and presence of DMPS SUVs (Figures 2 and 3) revealed that HSPB6 chaperone activity depends on the presence of lipid membranes. In order to better understand how different lipid types can influence the chaperone activity of HSPB6, and considering that α-syn can interact with different types of biological membranes, we generated membrane-mimetic SUVs with lipid composition that resemble either the outer leaflet of the plasma membrane (PM), the inner leaflet of the PM, the mitochondrial, the endoplasmic reticulum (ER), or the Golgi membranes (Figure 7A; Figure S5). Of note, these membrane-mimetic SUVs are composed of longer fatty acid chains (>18:0) compared to DMPS vesicles (14:0), conferring properties that are similar to the ones of physiological cellular and organelle membranes. We then studied whether these different types of lipids influence HSPB6 efficacy in preventing α-syn lipid-induced aggregation. The choice of these membrane-mimetic SUVs was driven by published data indicating that α-syn, which was initially identified as a protein enriched in the presynaptic terminal, where it associates with synaptic vesicles,71,72 can bind to a large variety of lipid membranes, including the plasma membrane, mitochondrial membranes, lipid rafts, and caveolae, as well as ER and Golgi membranes.73,74 Interaction with these lipid membranes, which occurs with different strengths based on their lipid composition, is important for α-syn physiological functions, but it is also tightly associated with α-syn pathological effects. For example, α-syn has a strong affinity for mitochondrial membranes75 and excessive binding of α-syn to these lipid membranes leads to neuronal toxicity.76 In addition, excessive association of α-syn with ER and Golgi impairs vesicular trafficking, promotes Golgi fragmentation and leads to the depletion of lysosomal enzymes in cellular and Drosophila models of PD.77 Finding that the efficacy of HSPB6 in inhibiting α-syn lipid-induced aggregation correlated with HSPB6 binding affinity to the lipid membranes suggests that it may protect different lipid membrane types from α-syn induced damage. In addition, it helps dissecting its mechanism of action. Currently, two mechanisms of action could be compatible with the results observed: competition with α-syn for binding to lipids (either as α-syn monomers or transient α-syn-lipid water-soluble complexes), and binding to α-syn monomers embedded into the lipids, thus shielding then from further oligomerization/aggregation. Future studies will need to address the question whether by binding to lipids HSPB6 changes their order, indirectly influencing α-syn binding to lipids.Figure 7 The binding affinity of HSPB6 for different lipid types influences the inhibition of α-syn lipid-induced aggregation

(A) Composition of membrane-mimetic SUVs resembling mitochondria membrane, the outer PM, the inner PM, the ER, or Golgi membranes.

(B) Binding affinity of α-syn (5 μM) for each membrane-mimetic SUV, expressed as fraction bound. Dissociation constant (Kd) values are as follows: 0.069 for mitochondria; 0.119 for outer PM; 0.082 for inner PM; 0.104 for ER; 0.104 for Golgi.

(C) Binding affinity of HSPB6 (5 μM) for each membrane-mimetic SUVs, expressed as fraction bound. Dissociation constant (Kd) values are as follows: 0.072 for mitochondria; 0.062 for outer PM; 0.045 for inner PM; 0.095 for ER; 0.088 for Golgi.

(D–H) Changes in ThT fluorescence intensity when monomeric α-syn (20 μM) is incubated with 100 μM of membrane-mimetic SUVs at pH 6.5°C and 30°C and in the absence (black) or presence of HSPBs (colored lines) at the following HSPB:α-syn ratios: 1:100 (0.2 μM:20 μM), 1:200 (0.1 μM:20 μM), 1:400 (0.05 μM: 20 μM), and 1:800 (0.025 μM: 20 μM).

(I) Correlation of the binding affinity of HSPB6 for lipids (Kd, x axis) with the half-time of aggregation at the 1:200 ratio (y axis), followed by analysis of the Pearson’s correlation coefficient (r): r = 0.85; p = 0.0375. (B–I) Data are shown as mean ± SEM. n = 3 independent experiments.

First, we measured the affinity of α-syn for the different membrane-mimetic SUVs and we calculated the fraction of α-syn bound to increasing concentrations of lipids. In agreement with previous findings,75 α-syn had the highest affinity for the mitochondrial membrane compared to the other membrane-mimetic SUVs (Figure 7B). In particular, α-syn showed the lowest affinity for SUVs mimicking the outer PM, and an intermediate affinity for SUVs mimicking the inner PM, Golgi, and ER (Figure 7B). Second, we measured the affinity of HSPB6 for the different membrane-mimetic SUVs. HSPB6 showed the highest binding affinity for SUVs mimicking the inner PM, followed by the outer PM and mitochondria (Figure 7C); the weakest interaction was detected for SUVs mimicking the composition of ER and Golgi membranes (Figure 7C).

We then evaluated the ability of HSPB6 to inhibit α-syn lipid-induced aggregation in presence of these different membrane-mimetic SUVs, using the ThT fluorescence assay and different HSPB6:α-syn ratios (Figures 7D–7H). At a 1:100 ratio, HSPB6 abrogated α-syn lipid-induced aggregation regardless the lipid types employed (Figures 7D–7H, compare green line with black line), while at the highest 1:800 ratio, HSPB6 lost its inhibitory effect under all conditions tested (Figure 7D, compare red line with black line). Instead, at the 1:200 and 1:400 ratios, HSPB6 delayed lipid-induced aggregation with efficiencies that varied based on the SUV composition (Figures 7D–7H, compare brown and orange lines with black line). We then calculated the normalized aggregation half-time (t1/2) at the 1:200 ratio and we correlated it (y axis) with the binding affinity for lipids (Kd, x axis), followed by analysis of the Pearson’s correlation coefficient (Figure 7I). This analysis revealed a significant correlation (r = 0.85, p value 0.0375) between the binding affinity of HSPB6 for lipids and its efficacy in preventing α-syn lipid-induced aggregation. The strongest inhibitory effect was exerted by HSPB6 in presence of SUVs that mimic the inner PM, followed by the outer PM and mitochondria. The lowest inhibitory effect of HSPB6 was observed when using SUVs that mimic the Golgi and the ER (Figure 7I), for which HSPB6 lipid-binding affinity was the lowest (Figure 7C). These data support the idea that HSPB6 is a lipid-dependent molecular chaperone.

Discussion

As α-syn aggregates are central to synucleopathies, tremendous efforts have been made to understand in detail the process of α-syn aggregation in order to identify effective ways to combat it. Achieving this goal is challenging because different paths lead to the formation of α-syn aggregates, including lipid-induced aggregation and α-syn liquid-liquid phase separation, followed by liquid-to-solid conversion.19 Moreover, environmental factors, such as pH, can accelerate specific steps of the aggregation process.6,7,69

Molecular chaperones, namely HSPs, have been for long considered as ideal candidates to prevent protein aggregation.78 Although much progress has been made in understanding molecular mechanisms of action of HSPs, and substantial evidence has been accumulated demonstrating their efficacy in blocking α-syn aggregation, the majority of the studies carried out to date have focused on a protein-centric view, so that several questions remain open on whether and how lipid membranes may influence the chaperone activity of HSPs.23,25,50,79 Here we have focused on the mammalian HSPB family, providing evidence that HSPBs evolved to block with multiple points of attack the kinetic network of microscopic processes leading to protein aggregation, with HSPB6 emerging as a lipid-dependent molecular chaperone.

In presence of lipid membranes, HSPB6 was by far more efficient than the other members of the family in preventing α-syn lipid-induced aggregation (Figures 2 and 3). By contrast, in absence of lipid membranes, HSPB6 showed a poor chaperone activity, whereas HSPB3, HSPB5 and HSPB7 were very efficient in preventing α-syn elongation, even at low concentrations (Figure 5) or at mildly acidic pH (Figure 6). Mechanistically, secondary nucleation consists of the fibril-dependent generation of new aggregates.80 These aggregates, in turn, elongate by addition of α-syn monomers and serve as a platform that amplifies α-syn aggregation.80 Previous reports showed that HSPBs can associate with fibrils during the aggregation process, delaying the process and affecting the morphology of the fibrils. The fibrils formed in presence of HSPBs were either similar but fewer in number (with HSPB5), shorter (with HSPB1 or HSPB2-HSPB3 complex) or no fibrils but compact structures formed (with HSPB8).28 Using a high concentration of α-syn monomers compared to that of preformed fibrils and progressively decreasing the concentration of the HSPBs tested enabled us to identify preferential binding of HSPB8 to monomeric versus fibrillar α-syn. This may help explain why HSPB8 changes the morphology of α-syn aggregates. Instead, HSPB3, HSPB5, and HSPB7, whose efficacy was maintained regardless of the concentration of monomeric free α-syn, preferentially bound to pre-formed fibrils (Figures 5 and 6). Given that also HSPB1 and HSPB5 bind to α-syn fibrils,26,30,50,54,68 binding to and coating the fibrils, yielding shorter or fewer fibrils, as previously reported,28 would represent a general mechanism by which HSPB combat aggregation. Instead, buffering free α-syn monomers and preventing their incorporation onto the fibril surface seems to be the preferred mechanism of action for HSPB8 (Figures 5 and 6), while acting at the α-syn protein-lipid interface appears as the action of choice for HSPB6 (Figures 2, 3, and 4). Intriguingly, HSPB8 shows the highest disorder and the highest propensity to undergo liquid-liquid phase separation, compared to the other HSPB members17 and was previously shown to prevent the aggregation of the RNA-binding protein FUS inside condensates by chaperoning its aggregation-prone RNA-binding domain.21 These observations suggest that HSPB8 could also co-condense with α-syn, in addition to interacting with the pre-formed fibrils as investigated in our work, preventing the maturation of α-syn condensates into amyloid aggregates. Concerning HSPB6, finding that its chaperone activity toward α-syn is lipid-dependent may explain why previous studies failed to identify any effect of HSPB6 on fibril morphology and only a minor effect on α-syn fibril formation,28 defining HSPB6 as a poor chaperone compared to the other members of the family.

We note that we still do not fully understand what regulates the lipid-dependent chaperone activity of HSPB6. At the highest HSPB:α-syn ratios, all HSPBs tested could efficiently delay α-syn-lipid induced aggregation, revealing a novel general mode of action for HSPBs at the protein-lipid interface. The ability of HSPBs to interact with lipids and prevent α-syn lipid-induced aggregation might be influenced by their degree of disorder. Of note, lipid binding is common among disordered proteins81 and HSPBs contain N-terminal and C-terminal intrinsically disordered domains.42 In addition, prion domains exhibit membrane binding propensities82; thus, the predicted prion-like domains in the HSPB protein sequence (http://plaac.wi.mit.edu/) may also enhance their binding affinity for lipids. Rather than a specific sequence, the structural heterogeneity of multiple coexisting conformations of HSPBs may be responsible for their binding to lipids. Binding to specific lipid types could transiently lock the HSPB molecules in an active conformation, similar to what happens upon post-translational modifications, influencing their chaperone activity. This inherent structural heterogeneity may explain why most HSPBs show efficient chaperone activity in absence of lipids, while HSPB6 binding to lipids would be required to unlock its chaperone power and prevent α-syn aggregation. The findings that the fluorescence polarization of DPH decreases in presence of HSPB6 or α-syn alone and further decreases when both proteins are co-incubated with SUVs (Figures 3 and S1) support the idea that HSPB6 directly interacts with lipid membranes. We cannot exclude the possibility that HSPB6 may bind to free lipids in equilibrium with the SUVs; in doing so, HSPB6 may decrease the formation of transient water-soluble lipid-α-syn complexes, ultimately reducing their incorporation into the lipid membranes. Alternatively, the changes in lipid membrane viscosity induced upon HSPB6 binding to them may change the local biophysical properties of the lipid membranes themselves, reducing the binding affinity of α-syn monomers. Another possible mechanism is that HSPB6 may bind to monomeric α-syn embedded into the lipid membranes, acting as a shield that would prevent aggregation. Yet, HSPB6 was shown to weakly and transiently bind to monomeric α-syn.28 Considering that the efficacy of HSPB6 in inhibiting α-syn lipid-induced aggregation correlates with its binding affinity for the lipids themselves (Figure 7), and in light of its poor chaperone ability in the absence of lipids and in the presence of excess monomeric α-syn (Figures 5 and 6),83,84 we favor the hypothesis that HSPB6 acts as a lipid-dependent molecular chaperone that interferes with α-syn embedding into the lipids. These data open the possibility that HSPB6 may preferentially inhibit the aggregation of fibril-forming proteins that bind to lipids.

This work informs on the importance of exploiting HSPB6 lipid-binding properties for the development of future therapeutic approaches in synucleopathies. HSPB6 is expressed throughout the brain, including the hippocampus, amygdala, cerebral cortex, and basal forebrain that degenerate in the advanced stages of PD,85 is upregulated in hippocampal neurons subjected to oxidative and hyperosmotic stress86 and exerts neuroprotective functions in vivo.87 Of note, HSPB1 and HSPB5 were also able to reduce α-syn lipid-induced aggregation, although with a lower efficacy compared to HSPB6. Considering that HSPB1 and HSPB5 are upregulated in the brain of patients affected by PD and other neurodegenerative diseases,88,89 we cannot exclude the possibility that their neuroprotective effects could be due, at least in part, also to the inhibition of lipid-induced protein aggregation.

Although it is still unclear where exactly α-syn lipid-induced aggregation occurs inside the cells, previous reports highlighted the mitochondria as the preferred subcellular site.76 The association of aggregated α-syn with mitochondria increases the production of toxic reactive oxygen species and impairs cellular respiration.76 In addition, transcellular propagation of α-syn aggregates, similar to prion protein aggregates, and impairment of membrane fusion events at synaptic terminals are well-established mechanisms contributing to synucleopathies.90 Of note, the membrane binding properties of α-syn are important for its internalization and spreading.91 HSPB6 interacts both with lipid vesicles mimicking mitochondrial and plasma membranes (Figure 7). Thus, increasing HSPB6 expression could not only protect mitochondria from α-syn-mediated toxicity but also prevent α-syn transcellular propagation.

In summary, our data support the view that protein aggregation takes place by a complex non-linear network of interconnected processes that are influenced not only by protein-protein interactions, but also by protein-lipid interactions. Recently, sharp and reversible transitions were described in the cell membranes of living neurons, further supporting their biological relevance for e.g., signal transduction and excitability.92 It is tempting to speculate that the direct interaction of the HSPBs with lipid membranes, supported by DPH fluorescence anisotropy measurements, and in agreement with previous studies,35,93,94,95,96 may contribute to stabilize the membranes upon stress conditions that influence membrane integrity and fluidity, such as heat shock and the presence of aggregation-prone proteins. Future studies will need to address to what extent the cytoprotective functions of HSPs are related to, or perhaps even dependent on, interactions with lipids. Our results indicate that HSPBs may have evolved to diversify their efficacy in a context-dependent manner, with some members becoming active only in presence of lipid membranes, and acting in different subcellular environments, both in solution and in phase-separated environments. These emerging properties may explain why, among the numerous molecular chaperones, small HSPs are highly represented in organisms exposed to extreme stressful conditions such as tardigrades,97 and why HSPBs are specifically upregulated in humans during aging, when the general protein homeostasis system declines.78

Limitations of the study

While our study provides valuable insights into the interactions between HSPBs and α-syn, several limitations must be acknowledged. Interaction with lipid vesicles: recent studies have shown that the binding of α-syn to model cell membranes that contain negatively charged lipids induces reversible structural changes that promote the formation of rod-like or disc-like lipid-protein particles.98 The small particles containing α-syn and lipids resulting from the fast and reversible rupture of the lipid vesicles can then be incorporated into the fibrils.98 This mechanism has been suggested to contribute to the formation of the pathological protein-lipid aggregates found in PD patients. In our work, we did not test whether/how HSPBs may influence the structural re-arrangements of the lipid vesicles into small particles. Future work should establish whether HSPBs affect the formation of specific discs or rods protein-lipid phases, thereby decreasing the frequency of unfavorable protein-protein interactions that facilitate amyloid fibril formation. Despite this limitation, we conclude that the interactions with lipids of HSPBs (especially HSPB6) can influence their anti-aggregation activity toward α-syn. Specificity of lipid interactions: the interactions of HSPBs, especially HSPB6, with a wider range of lipid membranes were not fully characterized. While we established that HSPB6 acts as a lipid-dependent molecular chaperone, further research is needed to determine the specific lipid compositions that most effectively modulate this chaperone activity. Understanding these specific interactions could provide deeper insights into the mechanisms by which HSPBs stabilize membranes and prevent protein aggregation. Environment-dependent activity: our study suggests that some HSPBs may become particularly active in the presence of lipid membranes, acting differently in various subcellular environments. However, the context-dependent efficacy of these molecular chaperones was not exhaustively investigated across all possible cellular conditions. Further studies will be needed to confirm these findings in diverse cellular and in vivo models to validate the proposed mechanisms. Mechanistic insights: while we propose that HSPBs might stabilize cell membranes and reduce amyloid fibril formation by influencing lipid-protein interactions, the corresponding molecular mechanisms remain to be fully elucidated. Detailed biophysical and structural studies will be required to characterize the precise ways in which HSPBs interact with lipid membranes and α-syn aggregates at the molecular level. In vivo relevance: since our findings are based on in vitro assays, their in vivo relevance will require further validation. Animal models and clinical studies will be necessary to confirm the protective roles of HSPBs, particularly HSPB6, in neurodegenerative diseases, including Parkinson’s disease. Comparative analysis: The study focused on a subset of HSPBs, finding that they have different relative efficacy in preventing α-syn aggregation. Future research should include a broader range of HSPBs to determine if other members of this family exhibit similar, or perhaps, superior anti-aggregation properties, as well as lipid-dependent chaperone activity.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Complete™, EDTA-free Protease Inhibitor Cocktail	Roche	11873580001	
IPTG	Invitrogen	15529–019	
Benzonase 250 U/mL	Sigma-Aldrich	E8263-5ku	
Arabinose	Lucigen	490234	
GsH	Sigma-Aldrich	G4251	
Thioflavin T UltraPure Grade	Eurogentec	AS-88306	
NaH2PO4, BioPerformance Certified >99.0%	Sigma-Aldrich	S6191	
Na2HPO4, ReagentPlus, >99.0%	Sigma-Aldrich	S0876	
NaN3, ReagentPlus, >99.5%	Sigma-Aldrich	S2002	
PC: L-α-phosphatidylcholine	Avanti Polar Lipids	840053P	
PE: L-α-phosphatidylethanolamine	Avanti Polar Lipids	840022P	
PI: L-α-phosphatidylinositol-4-phosphate	Avanti Polar Lipids	840045p	
PS: L-α-phosphatidylserine	Avanti Polar Lipids	840032P	
SM: Sphingomyelin	Avanti Polar Lipids	860062P	
chol: cholesterol	Avanti Polar Lipids	700100P	
DMPS (1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine) sodium salt	Avanti Polar Lipids	840033	
Diphenylhexatriene (DPH)	Sigma-Aldrich	SML0202	
HEPES	Sigma-Aldrich	H3375	
DTT	Sigma-Aldrich	D0632	
NaCl	Sigma-Aldrich	S3014	
KCl	Sigma-Aldrich	P3911	
Sodium Phosphate dibasic	Sigma-Aldrich	S9763	
Potassium Phosphate monobasic	Sigma-Aldrich	P0662	
α-synuclein	This paper	N/A	
HSPB3	This paper	N/A	
HSPB5	This paper	N/A	
HSPB6	This paper	N/A	
HSPB7	This paper	N/A	
HSPB8	This paper	N/A	
	
Bacterial and virus strains	
	
Escherichia coli strain BL21	Thermo Fisher	C607003	
	
Plasmid	
	
pET21a-HSPB3	Mymrikov et al.84	N/A	
pET23b-HSPB7	Mymrikov et al.84	N/A	
pET28b(+)-HSPB5	Mymrikov et al.84	N/A	
pUBS520-HSPB6	Mymrikov et al.84	N/A	
pGEX-4T-GST-HSPB8	Carra et al.99	N/A	
	
Software and algorithms	
	
Prism 8 (GraphPad)	GraphPad Software	https://www.graphpad.com/	
Daniel’s XL Toolbox	open-source add-in for Microsoft® Excel®	https://www.xltoolbox.net/	
	
Other	
	
Sephacryl S300 High-Prep 16/60 column	GE Healthcare	745515	
Superdex 200-pg column	GE Healthcare	28-9893-36	
Protino GST/4B Column	Macherey-Nagel	745515.5	
High-Trap Q HP column	GE Healthcare	17-1154-01	

Resource availability

Lead contact

Further information and requests for reagents should be directed to and will be fulfilled by the lead contact, serena.carra@unimore.it.

Materials availability

Cell lines generated in this study will be available upon reasonable request from the lead contact.

Data and code availability

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

• All data reported in this paper will be shared by the lead contact upon request.

• This paper does not report original code.

Method details

Protein production

α-syn wild-type and α-syn cysteine variant (N122C) were expressed and purified as previously described.80 Briefly, E. coli strain BL21 competent cells were transformed with pT7-7 plasmid coding wild type human α-syn. Protein expression was performed by addition of 1 mM isopropylβ-thiogalactopyranoside (IPTG, Merck) at 30°C for 4 h. Following purification, the purity of the aliquots was evaluated by SDS–PAGE and we used an extinction coefficient of 5600 M−1 cm−1 at 275 nm for protein concentration measurements. The protein solutions were aliquoted, flash frozen in liquid nitrogen and stored at −80°C, until required for use. Human small heat shock proteins were expressed in E. coli BL21(DE3) and purified as described elsewhere.84 Before use, proteins were dissolved or diluted in a phosphate buffer and dialyzed if necessary. Protein concentrations were calculated based on their absorbance values at 275 nm, with an extinction coefficient reported by the UniProt database.

Lipid vesicle preparation

Lipid powders (Avanti lipids) were dissolved following manufacturer’s instruction. 1,2-diacyl-sn-glycero-3-phospho-L-serine (DMPS; 14:0) lipid powder (840033, Avanti Lipids) was suspended in 20 mM phosphate buffer (NaH2PO4/Na2HPO4), pH 6.5, 0.01% NaN3 and stirred at 45°C (which corresponds to DMPS melting temperature) for 4 h as previously reported.7 Briefly, the solutions were frozen and thawed five times using dry ice and a water bath at 45°C. Lipid vesicles were prepared by sonication to avoid grains of segregated lipids (Bandelin, Sonopuls HD 2070, 3 × 5 min, 50% cycle, 10% maximum power) and centrifuged at 15,000 rpm for 30 min at 25°C (Medifuge Small Benchtop Centrifuge, Thermo Scientific). The sizes of the DMPS SUVs were checked using dynamic light scattering (Zetasizer Nano ZSP, Malvern Instruments, Malvern, UK) to consist of a distribution centered at 20 nm diameter. For the preparation of membrane-mimetic SUVs, lipid powders of the following lipids were used: L-α-phosphatidylcholine (840053P), referred to as phosphatidylcholine (PC; average fatty acid distribution: 33.3% 18:1, 30.6% 16:0, 16.5% 18:0, 3.1% 20:4, 4% other, 12.8% unknown), L-α-phosphatidylethanolamine (840022P), referred to as phosphatidylethanolamine (PE; average fatty acid distribution: 24.1% 18:1, 18.6% 20:4, 15.9% 18:0, 11.5% 22:6, 3.7% 16:0, unknown 21.1%, 5.4% other), L-α-phosphatidylinositol-4-phosphate (840045p), referred to as phosphatidylinositol (PI; average fatty acid distribution: 37.3% 18:0, 33.1% 20:4, 15.2% 18:1, 6.3% 16:0, 8.1% other), L-α-phosphatidylserine (840032P), referred to as phosphatidylserine (PS; average fatty acid distribution: 42% 18:0, 30% 18:1, 11% 22:6, 2% 20:4, 15% unknown), sphingomyelin (860062P), referred to as sphingomyelin (SM; average fatty acid distribution: 50% 18:0, 21% 24:1, 7% 22:0, 5% 24:0, 5% 20:0, 2% 16:0, 10% unknown) and cholesterol (700100P) (Avanti Polar Lipids) were suspended in 20 mM phosphate buffer (NaH2PO4/Na2HPO4), pH 6.5, 0.01% NaN3 and stirred at 45°C for 4 h. The membrane-mimetic SUVs were prepared by mixing in different combinations and percentage the different lipid types (as detailed in Figure 7A), as previously described.100 SUVs were used for the experiments immediately after dispersion and verification of their homogeneous size.

Site-directed mutagenesis

Vectors coding for phosphomimicking (S16D) and non-phosphorylatable (S16A) mutants of HSPB6 were generated using the vector coding for HSPB6 wild-type as template and the QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies).

Generation of an HSPB6 phosphorylation mimetic

Serine 16 of HSPB6 protein was mutated into a cysteine residue (S16C), followed by chemical conversion into a dehydroalanine (Dha) residue. 200 μM of the HSPB6 S16C protein (solubilized in 20 mM phosphate buffer, pH 7.4, was incubated on a shaker (500 rpm) with 5 M equivalents of methyl 2,5-dibromopentanoate 20 (Merck) for 48 h at 37°C, obtaining the intermediate product HSPB6-S16Dha. Excess methyl 2,5-dibromopentanoate was removed from HSPB6-S16Dha with 7k MWCO Zeba (Thermo Fisher) spin desalting columns. The conversion of the cysteine residue in position 16 to Dha of HSPB6-S16Dha was verified via LC-MS. Next, to add the phosphate group, 100 μL aliquots of 50 μM HSPB6-S16Dha in 20 nM sodium phosphate buffer, pH 7.4, were reacted batchwise (5 min intervals) with 30,000 M equivalents of sodium thiophosphate (pH 8.0, 690 mg/mL suspension, 5 × 5000 equivalents), for 24 h at 37°C and shaking at 500 rpm. Excess sodium thiophosphate was removed via 7k MWCO Zeba (ThermoFisher) spin desalting columns and reaction was verified via LC-MS.

Far-UV circular dichroism (CD) spectroscopy

CD samples were prepared by incubating 25 μM of HSPBs in 20 mM phosphate buffer, pH 6.5 or 5.5. Far-UV CD spectra were recorded on a JASCO J-810 spectrophotometer (JASCO UK, Ltd) equipped with a Peltier thermally controlled cuvette holder at 30°C (PIKE Technologies). Quartz cuvettes with path lengths of 1 mm were used and CD spectra were obtained by averaging three individual spectra recorded between wavelengths of 250 and 200 nm, with a bandwidth of 1 nm, a data pitch of 0.2 nm, a scanning speed of 50 nm/min, and a response time of 1 s. For each protein sample, the CD signal of the buffer used to solubilize the protein, or the signal of DMPS alone, was recorded and subtracted from the CD signal of the protein. The CD data was normalized in molar ellipticity per residue as per [q]res(deg cm2 dmol-1) = [q(mdeg) • mol weight > (g/mol)]/[(10 • number of res.· optical path(cm) • C (mg/cm3)].

Preparation of α-syn seed fibrils

α-syn seed fibrils were produced as described previously.69 500 μL samples of α-syn at concentrations from 500 to 800 μM were incubated in 20 mM phosphate buffer, pH 6.5, for 48 h at about 40°C and stirred at 1,500 rpm with a Teflon bar on an RCT Basic Heat Plate (IKA, Staufen, Germany). Fibrils were diluted to a monomer equivalent concentration of 200 μM, divided into aliquots, flash frozen in liquid N2 and stored at −80°C. For experiments at pH 6.5 the 200 μM fibril stock was sonicated for 30–60 s using a probe sonicator (Bandelin, Sonopuls HD 2070, Berlin, Germany) set at 10% maximum power and 50% cycle. For experiments at low pH with low seed, the 200 μM stock was diluted to 10 μM in water, sonicated 3 times for 5 s using 10% maximum power and 50% cycles.

Measurements of aggregation kinetics (ThT assay)

20 μM α-syn was incubated in the presence or absence of HSPBs and with 50 μM thioflavin T (ThT, Merck) and either preformed fibrils or DMPS vesicles at 37°C or 30°C, respectively.11 The change in the ThT fluorescence signal was monitored using a Fluostar Optima or Polarstar Omega fluorescence plate reader (BMG Labtech, Aylesbury, UK) in bottom reading mode under quiescent conditions. The concentrations of monomeric and fibrillar states of the protein were determined at the end of each aggregation experiment as previously described.11

Labeling α-syn and HSPB6

The N122C cysteine variant of α-syn80 and wild-type HSPB6 were buffer exchanged into phosphate buffer, pH 7.4, by use of P10 desalting columns packed with Sephadex G25 matrix (GE Healthcare). The protein was incubated overnight at 4°C on a rolling system with an excess of Alexa 488 with maleimide moieties (Thermofisher Scientific) at a molar ratio protein-to-dye of 1:1.5. To separate the labeled protein from the free dye, the labeling mixture was loaded onto a Superdex 200 16/600 (GE Healthcare) and eluted in 10 mM phosphate buffer, pH 7.4, at 20°C. The concentration of the labeled protein was estimated by the absorbance of the fluorophores, assuming a 1:1 labeling stoichiometry (Alexa 488: 72000 M−1 cm−1 at 495 nm).

Binding affinity assay

To determine the binding of α-syn or HSPB6 to lipid vesicles, we measured the change in the effective hydrodynamic radius (Rh) of the protein with increasing SUVs concentration. Measurements were done using 1 μM α-syn-S9C-AL488 and 4 μM α-syn or 1 μM HSPB6-AL488 and 4 μM HSPB6 at 25°C in 20 mM phosphate buffer pH 7.4. Progressive dilutions of SUV stock solution were prepared in 20 mM phosphate buffer pH 7.4. Samples were prepared with 3.5 μL of labeled protein, together with a varying concentration of the SUV of interest, and 3.5 μL of 20 mM phosphate buffer pH 7.4 in a 24-sample chip-plate. All the measurements were performed on a Fluidity One-W instrument (Fluidic Analytics, Cambridge, UK). Next, the change in the fraction of protein bound to lipid vesicles was fitted using:Fractionbound=X−XminXmax−Xmin,

where X = data point at specific lipid concentration (Rh); Xmin = Rh of free protein; Xmax = Rh of fully bound protein.

Analysis of the aggregation kinetics: Determination of the lipid-induced aggregation rate

The change in mass concentration of fibrils, M(t), was fitted using the single-step nucleation model described previously11:M(t)=KMk+m(0)n+1knbt22(KM+m(0))

where k+ is the elongation rate constant of fibrils from lipid vesicles, kn is the heterogeneous primary nucleation rate constant, n is the reaction order of the heterogeneous primary nucleation reaction relative to the free monomer, m, b is the total mass concentration of the protein bound to the lipid at 100% coverage (b=[DMPS]L, with L the stoichiometry) and KM is the Michaelis constant (fixed at 125 μM, as determined previously.7 This global analysis yields knk+ and n, for each variant. We then estimated the rate of aggregation of each variant on lipid vesicles, using AmyloFit101 to fit the kinetics to the model described above.

Quantification and statistical analysis

All statistical analysis were performed using one-way analysis of variance (ANOVA), followed by Bonferroni–Holm post hoc test using Daniel’s XL Toolbox or GraphPad Prism8 software. The data are shown as mean values of 3 independent experiments and error bars represent mean ± sem. Statistical significance was set at p < 0.05.

Supplemental information

Document S1. Figures S1–S5

Acknowledgments

We acknowledge Prof. J. Buchner for providing the vectors coding for HSPB3-5-6-7.84 We acknowledge Greta Šneiderienė for technical help with binding affinity measurements. S.C. acknowledges for funding 10.13039/501100021856 MUR (Departments of excellence 2018–2022; E91I18001480001 ), 10.13039/100009104 University of Modena and Reggio Emilia (FAR2020 ), 10.13039/501100002426 Telethon Foundation (GGP15001 and GMR22T1003 ), AriSLA Foundation (SUMOsolvable) and The Giovanni Armenise Harvard Foundation and AirAlzh (2022 AHA MCA ); M.V. acknowledges for funding the Center for Misfolding Diseases (10.13039/501100000735 University of Cambridge ). We acknowledge support from the Centre for Misfolding Diseases (University of Cambridge) and 10.13039/100014013 UKRI (grants 10059436 and 10061100 ).

Author contributions

S. Carra and M.V. conceived the study. V.S and T.T. performed the experiments and carried out the analyses with the assistance of R.S., S.P., and S. Chia. S. Carra and M.V. wrote the paper with input from all authors.

Declaration of interests

M.V. is a founder of WaveBreak Therapeutics (formerly Wren Therapeutics).

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110657.
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References

1 Yang Y. Shi Y. Schweighauser M. Zhang X. Kotecha A. Murzin A.G. Garringer H.J. Cullinane P.W. Saito Y. Foroud T. Structures of alpha-synuclein filaments from human brains with Lewy pathology Nature 610 2022 791 795 10.1038/s41586-022-05319-3 36108674
2 Sulzer D. Edwards R.H. The physiological role of alpha-synuclein and its relationship to Parkinson's Disease J. Neurochem. 150 2019 475 486 10.1111/jnc.14810 31269263
3 Sharma M. Burré J. alpha-Synuclein in synaptic function and dysfunction Trends Neurosci. 46 2023 153 166 10.1016/j.tins.2022.11.007 36567199
4 Lautenschlager J. Kaminski C.F. Kaminski Schierle G.S. alpha-Synuclein - Regulator of Exocytosis, Endocytosis, or Both? Trends Cell Biol. 27 2017 468 479 10.1016/j.tcb.2017.02.002 28259601
5 Eliezer D. Kutluay E. Bussell R. Jr. Browne G. Conformational properties of alpha-synuclein in its free and lipid-associated states J. Mol. Biol. 307 2001 1061 1073 10.1006/jmbi.2001.4538 11286556
6 Galvagnion C. Brown J.W.P. Ouberai M.M. Flagmeier P. Vendruscolo M. Buell A.K. Sparr E. Dobson C.M. Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of alpha-synuclein Proc. Natl. Acad. Sci. USA 113 2016 7065 7070 10.1073/pnas.1601899113 27298346
7 Galvagnion C. Buell A.K. Meisl G. Michaels T.C.T. Vendruscolo M. Knowles T.P.J. Dobson C.M. Lipid vesicles trigger alpha-synuclein aggregation by stimulating primary nucleation Nat. Chem. Biol. 11 2015 229 234 10.1038/nchembio.1750 25643172
8 Killinger B.A. Melki R. Brundin P. Kordower J.H. Endogenous alpha-synuclein monomers, oligomers and resulting pathology: let's talk about the lipids in the room NPJ Parkinsons Dis. 5 2019 23 10.1038/s41531-019-0095-3 31728405
9 Calabresi P. Mechelli A. Natale G. Volpicelli-Daley L. Di Lazzaro G. Ghiglieri V. Alpha-synuclein in Parkinson's disease and other synucleinopathies: from overt neurodegeneration back to early synaptic dysfunction Cell Death Dis. 14 2023 176 10.1038/s41419-023-05672-9 36859484
10 Lashuel H.A. Rethinking protein aggregation and drug discovery in neurodegenerative diseases: Why we need to embrace complexity? Curr. Opin. Chem. Biol. 64 2021 67 75 10.1016/j.cbpa.2021.05.006 34174698
11 Flagmeier P. Meisl G. Vendruscolo M. Knowles T.P.J. Dobson C.M. Buell A.K. Galvagnion C. Mutations associated with familial Parkinson's disease alter the initiation and amplification steps of alpha-synuclein aggregation Proc. Natl. Acad. Sci. USA 113 2016 10328 10333 10.1073/pnas.1604645113 27573854
12 Tsuang D. Leverenz J.B. Lopez O.L. Hamilton R.L. Bennett D.A. Schneider J.A. Buchman A.S. Larson E.B. Crane P.K. Kaye J.A. GBA mutations increase risk for Lewy body disease with and without Alzheimer disease pathology Neurology 79 2012 1944 1950 10.1212/WNL.0b013e3182735e9a 23035075
13 Alecu I. Bennett S.A.L. Dysregulated Lipid Metabolism and Its Role in alpha-Synucleinopathy in Parkinson's Disease Front. Neurosci. 13 2019 328 10.3389/fnins.2019.00328 31031582
14 Fanning S. Selkoe D. Dettmer U. Parkinson's disease: proteinopathy or lipidopathy? NPJ Parkinsons Dis. 6 2020 3 10.1038/s41531-019-0103-7 31909184
15 Cohen S.I.A. Vendruscolo M. Dobson C.M. Knowles T.P.J. From macroscopic measurements to microscopic mechanisms of protein aggregation J. Mol. Biol. 421 2012 160 171 10.1016/j.jmb.2012.02.031 22406275
16 Ray S. Singh N. Kumar R. Patel K. Pandey S. Datta D. Mahato J. Panigrahi R. Navalkar A. Mehra S. alpha-Synuclein aggregation nucleates through liquid-liquid phase separation Nat. Chem. 12 2020 705 716 10.1038/s41557-020-0465-9 32514159
17 Hardenberg M. Horvath A. Ambrus V. Fuxreiter M. Vendruscolo M. Widespread occurrence of the droplet state of proteins in the human proteome Proc. Natl. Acad. Sci. USA 117 2020 33254 33262 10.1073/pnas.2007670117 33318217
18 Hardenberg M.C. Sinnige T. Casford S. Dada S.T. Poudel C. Robinson E.A. Fuxreiter M. Kaminksi C.F. Kaminski Schierle G.S. Nollen E.A.A. Observation of an alpha-synuclein liquid droplet state and its maturation into Lewy body-like assemblies J. Mol. Cell Biol. 13 2021 282 294 10.1093/jmcb/mjaa075 33386842
19 Dada S.T. Hardenberg M.C. Toprakcioglu Z. Mrugalla L.K. Cali M.P. McKeon M.O. Klimont E. Michaels T.C.T. Knowles T.P.J. Vendruscolo M. Spontaneous nucleation and fast aggregate-dependent proliferation of alpha-synuclein aggregates within liquid condensates at neutral pH Proc. Natl. Acad. Sci. USA 120 2023 e2208792120 10.1073/pnas.2208792120
20 Arosio P. Michaels T.C.T. Linse S. Månsson C. Emanuelsson C. Presto J. Johansson J. Vendruscolo M. Dobson C.M. Knowles T.P.J. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation Nat. Commun. 7 2016 10948 10.1038/ncomms10948
21 Boczek E.E. Fursch J. Niedermeier M.L. Jawerth L. Jahnel M. Ruer-Gruss M. Kammer K.M. Heid P. Mediani L. Wang J. HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA-binding domain Elife 10 2021 e69377 10.7554/eLife.69377
22 Tao J. Berthet A. Citron Y.R. Tsiolaki P.L. Stanley R. Gestwicki J.E. Agard D.A. McConlogue L. Hsp70 chaperone blocks alpha-synuclein oligomer formation via a novel engagement mechanism J. Biol. Chem. 296 2021 100613 10.1016/j.jbc.2021.100613
23 Schneider M.M. Gautam S. Herling T.W. Andrzejewska E. Krainer G. Miller A.M. Trinkaus V.A. Peter Q.A.E. Ruggeri F.S. Vendruscolo M. The Hsc70 disaggregation machinery removes monomer units directly from alpha-synuclein fibril ends Nat. Commun. 12 2021 5999 10.1038/s41467-021-25966-w 34650037
24 Kappe G. Franck E. Verschuure P. Boelens W.C. Leunissen J.A. de Jong W.W. The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10 Cell Stress Chaperones 8 2003 53 61 10.1379/1466-1268(2003)8<53:thgecs>2.0.co;2 12820654
25 Cox D. Ecroyd H. The small heat shock proteins alphaB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of alpha-synuclein Cell Stress Chaperones 22 2017 589 600 10.1007/s12192-017-0785-x 28337642
26 Selig E.E. Zlatic C.O. Cox D. Mok Y.F. Gooley P.R. Ecroyd H. Griffin M.D.W. N- and C-terminal regions of alphaB-crystallin and Hsp27 mediate inhibition of amyloid nucleation, fibril binding, and fibril disaggregation J. Biol. Chem. 295 2020 9838 9854 10.1074/jbc.RA120.012748 32417755
27 Vicente Miranda H. Chegão A. Oliveira M.S. Fernandes Gomes B. Enguita F.J. Outeiro T.F. Hsp27 reduces glycation-induced toxicity and aggregation of alpha-synuclein Faseb. J. 34 2020 6718 6728 10.1096/fj.201902936R 32259355
28 Bruinsma I.B. Bruggink K.A. Kinast K. Versleijen A.A.M. Segers-Nolten I.M.J. Subramaniam V. Kuiperij H.B. Boelens W. de Waal R.M.W. Verbeek M.M. Inhibition of alpha-synuclein aggregation by small heat shock proteins Proteins 79 2011 2956 2967 10.1002/prot.23152 21905118
29 Cox D. Carver J.A. Ecroyd H. Preventing alpha-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins Biochim. Biophys. Acta 1842 2014 1830 1843 10.1016/j.bbadis.2014.06.024 24973551
30 Rekas A. Adda C.G. Andrew Aquilina J. Barnham K.J. Sunde M. Galatis D. Williamson N.A. Masters C.L. Anders R.F. Robinson C.V. Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity J. Mol. Biol. 340 2004 1167 1183 10.1016/j.jmb.2004.05.054 15236975
31 Renkawek K. Stege G.J. Bosman G.J. Dementia, gliosis and expression of the small heat shock proteins hsp27 and alpha B-crystallin in Parkinson's disease Neuroreport 10 1999 2273 2276 10.1097/00001756-199908020-00009 10439447
32 Seidel K. Vinet J. Dunnen W.F.A.d. Brunt E.R. Meister M. Boncoraglio A. Zijlstra M.P. Boddeke H.W.G.M. Rüb U. Kampinga H.H. Carra S. The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases Neuropathol. Appl. Neurobiol. 38 2012 39 53 10.1111/j.1365-2990.2011.01198.x 21696420
33 Chowdary T.K. Raman B. Ramakrishna T. Rao C.M. Interaction of mammalian Hsp22 with lipid membranes Biochem. J. 401 2007 437 445 10.1042/BJ20061046 17020537
34 Lopez V. Cauvi D.M. Arispe N. De Maio A. Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes Cell Stress Chaperones 21 2016 609 616 10.1007/s12192-016-0685-5 27075190
35 De Maio A. Cauvi D.M. Capone R. Bello I. Egberts W.V. Arispe N. Boelens W. The small heat shock proteins, HSPB1 and HSPB5, interact differently with lipid membranes Cell Stress Chaperones 24 2019 947 956 10.1007/s12192-019-01021-y 31338686
36 Auluck P.K. Caraveo G. Lindquist S. alpha-Synuclein: membrane interactions and toxicity in Parkinson's disease Annu. Rev. Cell Dev. Biol. 26 2010 211 233 10.1146/annurev.cellbio.042308.113313 20500090
37 Dores-Silva P.R. Cauvi D.M. Coto A.L.S. Silva N.S.M. Borges J.C. De Maio A. Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes Cell Stress Chaperones 26 2021 671 684 10.1007/s12192-021-01210-8 34003451
38 Balogi Z. Multhoff G. Jensen T.K. Lloyd-Evans E. Yamashima T. Jäättelä M. Harwood J.L. Vígh L. Hsp70 interactions with membrane lipids regulate cellular functions in health and disease Prog. Lipid Res. 74 2019 18 30 10.1016/j.plipres.2019.01.004 30710597
39 Tsvetkova N.M. Horváth I. Török Z. Wolkers W.F. Balogi Z. Shigapova N. Crowe L.M. Tablin F. Vierling E. Crowe J.H. Vigh L. Small heat-shock proteins regulate membrane lipid polymorphism Proc. Natl. Acad. Sci. USA 99 2002 13504 13509 10.1073/pnas.192468399 12368478
40 D'Anneo A. Bavisotto C.C. Gammazza A.M. Paladino L. Carlisi D. Cappello F. de Macario E.C. Macario A.J.L. Lauricella M. Lipid chaperones and associated diseases: a group of chaperonopathies defining a new nosological entity with implications for medical research and practice Cell Stress Chaperones 25 2020 805 820 10.1007/s12192-020-01153-6 32856199
41 Sciacca M.F. Lolicato F. Tempra C. Scollo F. Sahoo B.R. Watson M.D. García-Viñuales S. Milardi D. Raudino A. Lee J.C. Lipid-Chaperone Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins ACS Chem. Neurosci. 11 2020 4336 4350 10.1021/acschemneuro.0c00588 33269918
42 Carra S. Alberti S. Benesch J.L.P. Boelens W. Buchner J. Carver J.A. Cecconi C. Ecroyd H. Gusev N. Hightower L.E. Small heat shock proteins: multifaceted proteins with important implications for life Cell Stress Chaperones 24 2019 295 308 10.1007/s12192-019-00979-z 30758704
43 Sudnitsyna M.V. Mymrikov E.V. Seit-Nebi A.S. Gusev N.B. The role of intrinsically disordered regions in the structure and functioning of small heat shock proteins Curr. Protein Pept. Sci. 13 2012 76 85 10.2174/138920312799277875 22044147
44 Boelens W.C. Structural aspects of the human small heat shock proteins related to their functional activities Cell Stress Chaperones 25 2020 581 591 10.1007/s12192-020-01093-1 32253739
45 Weeks S.D. Baranova E.V. Heirbaut M. Beelen S. Shkumatov A.V. Gusev N.B. Strelkov S.V. Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6 J. Struct. Biol. 185 2014 342 354 10.1016/j.jsb.2013.12.009 24382496
46 den Engelsman J. Boros S. Dankers P.Y.W. Kamps B. Vree Egberts W.T. Böde C.S. Lane L.A. Aquilina J.A. Benesch J.L.P. Robinson C.V. The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio J. Mol. Biol. 393 2009 1022 1032 10.1016/j.jmb.2009.08.052 19715703
47 Alderson T.R. Ying J. Bax A. Benesch J.L.P. Baldwin A.J. Conditional Disorder in Small Heat-shock Proteins J. Mol. Biol. 432 2020 3033 3049 10.1016/j.jmb.2020.02.003 32081587
48 Janowska M.K. Baughman H.E.R. Woods C.N. Klevit R.E. Mechanisms of Small Heat Shock Proteins Cold Spring Harbor Perspect. Biol. 11 2019 a034025 10.1101/cshperspect.a034025
49 Bendifallah M. Redeker V. Monsellier E. Bousset L. Bellande T. Melki R. Interaction of the chaperones alpha B-crystallin and CHIP with fibrillar alpha-synuclein: Effects on internalization by cells and identification of interacting interfaces Biochem. Biophys. Res. Commun. 527 2020 760 769 10.1016/j.bbrc.2020.04.091 32430178
50 Cox D. Whiten D.R. Brown J.W.P. Horrocks M.H. San Gil R. Dobson C.M. Klenerman D. van Oijen A.M. Ecroyd H. The small heat shock protein Hsp27 binds alpha-synuclein fibrils, preventing elongation and cytotoxicity J. Biol. Chem. 293 2018 4486 4497 10.1074/jbc.M117.813865 29382725
51 Jia C. Ma X. Liu Z. Gu J. Zhang X. Li D. Zhang S. Different Heat Shock Proteins Bind alpha-Synuclein With Distinct Mechanisms and Synergistically Prevent Its Amyloid Aggregation Front. Neurosci. 13 2019 1124 10.3389/fnins.2019.01124 31749672
52 Lu S.Z. Guo Y.S. Liang P.Z. Zhang S.Z. Yin S. Yin Y.Q. Wang X.M. Ding F. Gu X.S. Zhou J.W. Suppression of astrocytic autophagy by alphaB-crystallin contributes to alpha-synuclein inclusion formation Transl. Neurodegener. 8 2019 3 10.1186/s40035-018-0143-7 30675347
53 Outeiro T.F. Klucken J. Strathearn K.E. Liu F. Nguyen P. Rochet J.C. Hyman B.T. McLean P.J. Small heat shock proteins protect against alpha-synuclein-induced toxicity and aggregation Biochem. Biophys. Res. Commun. 351 2006 631 638 10.1016/j.bbrc.2006.10.085 17081499
54 Waudby C.A. Knowles T.P.J. Devlin G.L. Skepper J.N. Ecroyd H. Carver J.A. Welland M.E. Christodoulou J. Dobson C.M. Meehan S. The interaction of alphaB-crystallin with mature alpha-synuclein amyloid fibrils inhibits their elongation Biophys. J. 98 2010 843 851 10.1016/j.bpj.2009.10.056 20197038
55 Perni M. Flagmeier P. Limbocker R. Cascella R. Aprile F.A. Galvagnion C. Heller G.T. Meisl G. Chen S.W. Kumita J.R. Multistep Inhibition of alpha-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine ACS Chem. Biol. 13 2018 2308 2319 10.1021/acschembio.8b00466 29953201
56 Buell A.K. Dobson C.M. Knowles T.P.J. The physical chemistry of the amyloid phenomenon: thermodynamics and kinetics of filamentous protein aggregation Essays Biochem. 56 2014 11 39 10.1042/bse0560011 25131584
57 Sinning A. Hübner C.A. Minireview: pH and synaptic transmission FEBS Lett. 587 2013 1923 1928 10.1016/j.febslet.2013.04.045 23669358
58 Fusco G. Chen S.W. Williamson P.T.F. Cascella R. Perni M. Jarvis J.A. Cecchi C. Vendruscolo M. Chiti F. Cremades N. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers Science 358 2017 1440 1443 10.1126/science.aan6160 29242346
59 Demchenko A.P. Mély Y. Duportail G. Klymchenko A.S. Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes Biophys. J. 96 2009 3461 3470 10.1016/j.bpj.2009.02.012 19413953
60 Kato K. Goto S. Inaguma Y. Hasegawa K. Morishita R. Asano T. Purification and characterization of a 20-kDa protein that is highly homologous to alpha B crystallin J. Biol. Chem. 269 1994 15302 15309 8195168
61 Edwards H.V. Scott J.D. Baillie G.S. PKA phosphorylation of the small heat-shock protein Hsp20 enhances its cardioprotective effects Biochem. Soc. Trans. 40 2012 210 214 10.1042/BST20110673 22260692
62 Chernik I.S. Seit-Nebi A.S. Marston S.B. Gusev N.B. Small heat shock protein Hsp20 (HspB6) as a partner of 14-3-3gamma Mol. Cell. Biochem. 295 2007 9 17 10.1007/s11010-006-9266-8 17109079
63 Dreiza C.M. Brophy C.M. Komalavilas P. Furnish E.J. Joshi L. Pallero M.A. Murphy-Ullrich J.E. von Rechenberg M. Ho Y.s.J. Richardson B. Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics FASEB J. 19 2005 261 263 10.1096/fj.04-2911fje 15598710
64 Underwood R. Gannon M. Pathak A. Kapa N. Chandra S. Klop A. Yacoubian T.A. 14-3-3 mitigates alpha-synuclein aggregation and toxicity in the in vivo preformed fibril model Acta Neuropathol. Commun. 9 2021 13 10.1186/s40478-020-01110-5 33413679
65 Jakubec M. Bariås E. Furse S. Govasli M.L. George V. Turcu D. Iashchishyn I.A. Morozova-Roche L.A. Halskau Ø. Cholesterol-containing lipid nanodiscs promote an alpha-synuclein binding mode that accelerates oligomerization FEBS J. 288 2021 1887 1905 10.1111/febs.15551 32892498
66 Sluchanko N.N. Beelen S. Kulikova A.A. Weeks S.D. Antson A.A. Gusev N.B. Strelkov S.V. Structural Basis for the Interaction of a Human Small Heat Shock Protein with the 14-3-3 Universal Signaling Regulator Structure 25 2017 305 316 10.1016/j.str.2016.12.005 28089448
67 Sluchanko N.N. Sudnitsyna M.V. Chernik I.S. Seit-Nebi A.S. Gusev N.B. Phosphomimicking mutations of human 14-3-3zeta affect its interaction with tau protein and small heat shock protein HspB6 Arch. Biochem. Biophys. 506 2011 24 34 10.1016/j.abb.2010.11.003 21081103
68 Shammas S.L. Waudby C.A. Wang S. Buell A.K. Knowles T.P.J. Ecroyd H. Welland M.E. Carver J.A. Dobson C.M. Meehan S. Binding of the molecular chaperone alphaB-crystallin to Abeta amyloid fibrils inhibits fibril elongation Biophys. J. 101 2011 1681 1689 10.1016/j.bpj.2011.07.056 21961594
69 Buell A.K. Galvagnion C. Gaspar R. Sparr E. Vendruscolo M. Knowles T.P.J. Linse S. Dobson C.M. Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation Proc. Natl. Acad. Sci. USA 111 2014 7671 7676 10.1073/pnas.1315346111 24817693
70 Rajagopal P. Tse E. Borst A.J. Delbecq S.P. Shi L. Southworth D.R. Klevit R.E. A conserved histidine modulates HSPB5 structure to trigger chaperone activity in response to stress-related acidosis Elife 4 2015 e07304 10.7554/eLife.07304
71 Maroteaux L. Campanelli J.T. Scheller R.H. Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal J. Neurosci. 8 1988 2804 2815 10.1523/JNEUROSCI.08-08-02804.1988 3411354
72 Kahle P.J. Neumann M. Ozmen L. Muller V. Jacobsen H. Schindzielorz A. Okochi M. Leimer U. van Der Putten H. Probst A. Subcellular localization of wild-type and Parkinson's disease-associated mutant alpha -synuclein in human and transgenic mouse brain J. Neurosci. 20 2000 6365 6373 10.1523/JNEUROSCI.20-17-06365.2000 10964942
73 Snead D. Eliezer D. Alpha-synuclein function and dysfunction on cellular membranes Exp. Neurobiol. 23 2014 292 313 10.5607/en.2014.23.4.292 25548530
74 Miraglia F. Ricci A. Rota L. Colla E. Subcellular localization of alpha-synuclein aggregates and their interaction with membranes Neural Regen. Res. 13 2018 1136 1144 10.4103/1673-5374.235013 30028312
75 Nakamura K. Nemani V.M. Wallender E.K. Kaehlcke K. Ott M. Edwards R.H. Optical reporters for the conformation of alpha-synuclein reveal a specific interaction with mitochondria J. Neurosci. 28 2008 12305 12317 10.1523/JNEUROSCI.3088-08.2008 19020024
76 Choi M.L. Chappard A. Singh B.P. Maclachlan C. Rodrigues M. Fedotova E.I. Berezhnov A.V. De S. Peddie C.J. Athauda D. Author Correction: Pathological structural conversion of alpha-synuclein at the mitochondria induces neuronal toxicity Nat. Neurosci. 25 2022 1582 10.1038/s41593-022-01206-2 36261654
77 Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models Science 313 2006 324 328 10.1126/science.1129462 16794039
78 Hipp M.S. Kasturi P. Hartl F.U. The proteostasis network and its decline in ageing Nat. Rev. Mol. Cell Biol. 20 2019 421 435 10.1038/s41580-019-0101-y 30733602
79 Gao X. Carroni M. Nussbaum-Krammer C. Mogk A. Nillegoda N.B. Szlachcic A. Guilbride D.L. Saibil H.R. Mayer M.P. Bukau B. Human Hsp70 Disaggregase Reverses Parkinson's-Linked alpha-Synuclein Amyloid Fibrils Mol. Cell 59 2015 781 793 10.1016/j.molcel.2015.07.012 26300264
80 Gaspar R. Meisl G. Buell A.K. Young L. Kaminski C.F. Knowles T.P.J. Sparr E. Linse S. Secondary nucleation of monomers on fibril surface dominates alpha-synuclein aggregation and provides autocatalytic amyloid amplification Q. Rev. Biophys. 50 2017 e6 10.1017/S0033583516000172 29233218
81 Deryusheva E. Nemashkalova E. Galloux M. Richard C.A. Eléouët J.F. Kovacs D. Van Belle K. Tompa P. Uversky V. Permyakov S. Does Intrinsic Disorder in Proteins Favor Their Interaction with Lipids? Proteomics 19 2019 e1800098 10.1002/pmic.201800098
82 Overduin M. Wille H. Westaway D. Multisite interactions of prions with membranes and native nanodiscs Chem. Phys. Lipids 236 2021 105063 10.1016/j.chemphyslip.2021.105063
83 van de Klundert F.A. Smulders R.H. Gijsen M.L. Lindner R.A. Jaenicke R. Carver J.A. de Jong W.W. The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone Eur. J. Biochem. 258 1998 1014 1021 10.1046/j.1432-1327.1998.2581014.x 9990320
84 Mymrikov E.V. Daake M. Richter B. Haslbeck M. Buchner J. The Chaperone Activity and Substrate Spectrum of Human Small Heat Shock Proteins J. Biol. Chem. 292 2017 672 684 10.1074/jbc.M116.760413 27909051
85 Zhou C. Wang L. Cheng W. Lv J. Guan X. Guo T. Wu J. Zhang W. Gao T. Liu X. Two distinct trajectories of clinical and neurodegeneration events in Parkinson's disease NPJ Parkinsons Dis. 9 2023 111 10.1038/s41531-023-00556-3 37443179
86 Bartelt-Kirbach B. Golenhofen N. Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons Cell Stress Chaperones 19 2014 145 153 10.1007/s12192-013-0452-9 23959629
87 Qi A.Q. Zhang Y.H. Qi Q.D. Liu Y.H. Zhu J.L. Overexpressed HspB6 Underlines a Novel Inhibitory Role in Kainic Acid-Induced Epileptic Seizure in Rats by Activating the cAMP-PKA Pathway Cell. Mol. Neurobiol. 39 2019 111 122 10.1007/s10571-018-0637-y 30511325
88 Vendredy L. Adriaenssens E. Timmerman V. Small heat shock proteins in neurodegenerative diseases Cell Stress Chaperones 25 2020 679 699 10.1007/s12192-020-01101-4 32323160
89 Navarro-Zaragoza J. Cuenca-Bermejo L. Almela P. Laorden M.L. Herrero M.T. Could Small Heat Shock Protein HSP27 Be a First-Line Target for Preventing Protein Aggregation in Parkinson's Disease? Int. J. Mol. Sci. 22 2021 3038 10.3390/ijms22063038
90 Melki R. Alpha-synuclein and the prion hypothesis in Parkinson's disease Rev. Neurol. 174 2018 644 652 10.1016/j.neurol.2018.08.002 30201422
91 Masaracchia C. Hnida M. Gerhardt E. Lopes da Fonseca T. Villar-Pique A. Branco T. Stahlberg M.A. Dean C. Fernández C.O. Milosevic I. Outeiro T.F. Membrane binding, internalization, and sorting of alpha-synuclein in the cell Acta Neuropathol. Commun. 6 2018 79 10.1186/s40478-018-0578-1 30107856
92 Fedosejevs C.S. Schneider M.F. Sharp, localized phase transitions in single neuronal cells Proc. Natl. Acad. Sci. USA 119 2022 e2117521119 10.1073/pnas.2117521119
93 De Maio A. Hightower L. The interaction of heat shock proteins with cellular membranes: a historical perspective Cell Stress Chaperones 26 2021 769 783 10.1007/s12192-021-01228-y 34478113
94 Csoboz B. Gombos I. Kota Z. Dukic B. Klement E. Varga-Zsiros V. Lipinszki Z. Pali T. Vigh L. Torok Z. The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes Int. J. Mol. Sci. 23 2022 7317 10.3390/ijms23137317
95 Crul T. Csoboz B. Gombos I. Marton A. Peter M. Balogh G. Vizler C. Szente L. Vigh L. Modulation of Plasma Membrane Composition and Microdomain Organization Impairs Heat Shock Protein Expression in B16-F10 Mouse Melanoma Cells Cells 9 2020 951 10.3390/cells9040951
96 Torok Z. Crul T. Maresca B. Schutz G.J. Viana F. Dindia L. Piotto S. Brameshuber M. Balogh G. Peter M. Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications Biochim. Biophys. Acta 1838 2014 1594 1618 10.1016/j.bbamem.2013.12.015 24374314
97 Hibshman J.D. Carra S. Goldstein B. Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation Commun. Biol. 6 2023 121 10.1038/s42003-023-04512-y 36717706
98 Galvagnion C. Barclay A. Makasewicz K. Marlet F.R. Moulin M. Devos J.M. Linse S. Martel A. Porcar L. Sparr E. Structural characterisation of alpha-synuclein-membrane interactions and the resulting aggregation using small angle scattering Phys. Chem. Chem. Phys. 26 2024 10998 11013 10.1039/d3cp05928f 38526443
99 Carra S. Sivilotti M. Chávez Zobel A.T. Lambert H. Landry J. HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells Hum. Mol. Genet. 14 2005 1659 1669 10.1093/hmg/ddi174 15879436
100 Baumann K.N. Šneiderienė G. Sanguanini M. Schneider M. Rimon O. González Díaz A. Greer H. Thacker D. Linse S. Knowles T.P.J. Vendruscolo M. A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Abeta(42) Aggregation ACS Chem. Neurosci. 14 2023 323 329 10.1021/acschemneuro.2c00765 36574473
101 Meisl G. Kirkegaard J.B. Arosio P. Michaels T.C.T. Vendruscolo M. Dobson C.M. Linse S. Knowles T.P.J. Molecular mechanisms of protein aggregation from global fitting of kinetic models Nat. Protoc. 11 2016 252 272 10.1038/nprot.2016.010 26741409
