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. 2016 Feb:83:433-41.
doi: 10.1016/j.ijbiomac.2015.10.089. Epub 2015 Nov 19.

The C-terminal α-helices of mammalian Hsc70 play a critical role in the stabilization of α-synuclein binding and inhibition of aggregation

Ali Chaari  1 , David Eliezer  2 , Moncef Ladjimi  3
Affiliations

The C-terminal α-helices of mammalian Hsc70 play a critical role in the stabilization of α-synuclein binding and inhibition of aggregation

Ali Chaari et al. Int J Biol Macromol. 2016 Feb.

Abstract

Protein misfolding, followed by aggregation and amyloid formation is an underlying pathological hallmark in a number of prevalent diseases, including Parkinson's (PD), Alzheimer's (AD) and Type 2 diabetes (T2D). In the case of PD, the aggregation of α-synuclein protein (α-syn) has been shown to be highly cytotoxic and to play a key role in the death of dopaminergic cells. Thus, inhibition of the aggregation process may be considered as an attractive avenue for therapeutic intervention. In this respect, molecular chaperones, known to promote proper folding of proteins, are able to inhibit protein aggregation thus preventing amyloid formation. In this work, the effect of the constitutively expressed chaperone Hsc70 and its various domains on α-syn aggregation have been investigated using different approaches. The results show that the C-terminal domain alone (residues 386-646) is as efficient in inhibiting α-syn aggregation as the entire Hsc70 protein, by increasing the lag phase for α-syn oligomeric nucleus formation, suggesting that the chaperone interacts with and stabilizes α-syn monomers and/or small aggregates. Deletion of the C-terminal helices (residues 510-646), which are known to play the role of a lid locking target peptide ligands in the peptide-binding site of the chaperone, strongly reduced the efficiency of inhibition of α-syn aggregation indicating that these helices play an essential in stabilizing the interaction between Hsc70 and α-syn. Furthermore, the effects of Hsc70 and its structural domains on aggregation appear to correlate with those on cytotoxicity, by reducing the fraction of α-syn toxic species to various degrees. Together these results suggest a mechanism in which inhibition of synuclein aggregation is the result of monomeric synuclein binding to the chaperone as any monomeric target unfolded protein or peptide binding to the chaperone.

Keywords: Aggregation; Alpha synuclein; Amyloid fibril; Hsc70 molecular chaperone.

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Figures

Figure 1
Figure 1. Representation of Hsc70 and its respective structural domains
Top: Structure of DnaK based on the PDB file 2KHO [37] 19. Bottom: Schematic representation of Hsc70 and its structural domains represented by different colors.
Figure 2
Figure 2. Analysis of purified α-syn, Hsc70 and its structural domains by size exclusion chromatography and SDS-PAGE (inset)
5 mM of these proteins were loaded on a superdex 75 column using high performance liquid chromatography (HPLC) and eluted as described under « Materials and methods ». Inset: 15 mg of each purified protein were analyzed by 12% SDS-PAGE as described under « Materials and methods ».
Figure 3
Figure 3. Aggregation of α-syn
A) Time course of aggregation monitored by ThT fluorescence (10 μM, red; 35 μM, orange; 70 μM, blue and 100 μM, black). The time-dependence of ThT fluorescence was fitted to a sigmoidal growth model as described under « Materials and methods ». ThT fluorescence results are the means of three independent experiments. B) AFM images of 70 μM α-syn at 0 h (sample 1), and 52h (sample 2). C) DLS histograms of 70 μM α-syn at 0 h (sample 1) and 52h (sample 2).
Figure 4
Figure 4. Aggregation of α-syn in presence of Hsc70
A) Time course of α-syn aggregation monitored by ThT fluorescence in the absence (black) and presence of increasing amounts of Hsc70 (0.7 μM, bleu; 1.4 μM, brown; 3.5 μM, red and 7 μM, violet). The time-dependence of ThT fluorescence was fitted to a sigmoidal growth model as described under « Materials and methods ». Inset: percentage of Inhibition of α-syn aggregation at increasing ratios of [Hsc70]/[α-syn]. This % of inhibition was calculated at 52h taking the fluorescence of -syn in absence of Hsc70 as 0% of aggregation. ThT fluorescence results are the means of three independent experiments. B) AFM images of 70 μM α-syn at 0 h (sample 1), and 52h in presence of 7μM of Hsc70 (sample 2). C) DLS histograms of 70 μM α-syn at 0 h (sample 1) and 52h in presence of 7μM of Hsc70 (sample 2).
Figure 5
Figure 5. The inhibition of α-syn aggregation by structural domains of Hsc70
A) The time dependence of amyloid inhibition by Hsc70 and its structural domains. B) Extent of inhibition (%) of α-syn aggregation by Hsc70 and its structural domains (% calculated with respect to the ThT fluorescence of α-syn at 52h) at increasing ratios of [Hsc70]/[α-syn]. Inset: Percentage of Inhibition of α-syn aggregation at a constant α-syn concentration (70 μM) and increasing concentrations of Hsc70 and its respective structural domains at 52h of incubation. This inhibition was fitted using the hyperbolic equation Y = Bmax*X/(Kd + X). Where Bmax is the maximum specific binding in the same units as Y. It is the specific binding extrapolated to very high concentrations of [Hsc70], and so its value is almost always higher than any specific binding measured in the experiment. Kd is the equilibrium binding constant, in the same units as X. It is the chaperone concentration needed to achieve a half-maximum binding at equilibrium. ThT fluorescence results are the means of three independent experiments. C) DLS analysis of α-syn in presence and absence of Hsc70 and its structural domains. The histograms represent the mass percentage of aggregates species.
Figure 6
Figure 6. Cell viability assays
Viability of SHSY5Y neuronal cells upon exposure to soluble α-syn at 0h (grey histogram) or α-syn aggregates in the absence and presence of Hsc70 and its domains sampled at 52h (yellow histogram). The final protein concentration within the culture medium was 1 μM. The cells were incubated in the absence and presence of Hsc70 chaperone and its structural domains for 24 h. Cell viability is expressed as the percentage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction using cells treated with the same volume of buffer as a reference (100% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction). The values (averages ± S.D.) are obtained from three independent experiments.
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