Introduction
Parkinson's disease (PD) is a progressive neurodegenerative movement disorder
distinguished neuropathologically by the presence of Lewy bodies and Lewy
neurites—proteinaceous cytoplasmic inclusions—in dopaminergic
neurons of the substantia nigra (1). Electron microscopy shows these inclusions to be composed of
amyloid-like fibrillar material, the major component of which is the 140-residue,
intrinsically disordered protein α-synuclein
(αSyn) (2).
Recombinant αSyn has been found to form aggregates
in vitro with all the characteristics of amyloid fibrils associated with
diseased states (3). Although most cases of
PD are idiopathic, a small number of familial forms have been identified including
the A53T variant, in which aggregation is significantly accelerated relative to the
wild-type (WT) (4), thus implicating
αSyn aggregation in the etiology of PD. As
αSyn knockout mice do not exhibit PD-like symptoms, it
can be concluded that PD results from a pathological gain of function (i.e.,
aggregate toxicity) rather than the loss of normal αSyn
function (5).
The aggregation mechanism itself, in common with other amyloid systems, is a
nucleated process in which there is slow and initially unfavorable association of
monomers into a structure competent to elongate into full-length fibrils (6). An important experimental consequence of
such a nucleation-polymerization mechanism is the phenomenon of seeding, in which a
substoichiometric quantity of preformed fibrils can greatly accelerate the
aggregation reaction by bypassing the nucleation process. The aggregation kinetics
of seeded reactions are therefore determined only by the elongation and
fragmentation rates, and, in the initial rate approximation, by the elongation rate
alone (7).
αSyn aggregation may be suppressed by the molecular
chaperone Hsp70, and biophysical investigations have shown that this
inhibition occurs via binding to prefibrillar species rather than monomeric
αSyn (8).
Another molecular chaperone that inhibits αSyn aggregation
is αB-crystallin, a 175-residue protein ubiquitous
in mammalian tissue (9). Early work showed
that αB-crystallin is a member of the family of small heat
shock proteins (sHsps), and is upregulated in response to a range of stress stimuli
and clinical disorders including Alzheimer's disease, transmissible spongiform
encephalopathies, dementia with Lewy bodies, and Parkinson's disease (10). As a sHsp,
αB-crystallin is a molecular chaperone and has been
demonstrated to suppress thermally induced aggregation of
β- and γ-crystallins, suggesting
that it acts as a significant protective mechanism against cataract formation in the
eye lens (11); the latter is associated
with aggregation and has been shown in some cases to involve amyloid formation
(12).
αB-Crystallin also inhibits the aggregation of several
other proteins in vitro, including the amyloid-β
peptides, β2-microglobulin, and insulin (13,14). There are, however, conflicting
reports as to whether such inhibition is neuroprotective (15,16).
The sequence of αB-crystallin has amphipathic character, and
contains a hydrophobic N-terminal domain, a central
α-crystallin domain (common to all sHsps), and a
hydrophilic and flexible 12-residue C-terminal extension (9). The amphipathic nature of the sequence is believed to
be crucial to its chaperone function, enabling
αB-crystallin to bind to exposed hydrophobic patches
characteristic of misfolded states (17).
The hydrophilic and highly flexible C-terminal extension, as characterized by
solution state nuclear magnetic resonance (NMR) (18), is also essential to efficient chaperone activity, with an
important role in solubilizing complexes of the chaperone with misfolded and
aggregated species (10). No crystal
structure of αB-crystallin has yet been determined;
comparison of the α-crystallin domain with
structures of other sHsps, however, suggests that the central domain will adopt a
β-sandwich structure (19). A major barrier to structural characterization of
αB-crystallin, and certainly to its crystallization, is
the self-association of monomers into large, polydispersed assemblies (typically
24–33mers), with molecular masses varying from 300 kDa to >1 MDa
(20). Studies of the quaternary
structure of the protein by cryo-electron microscopy reveal spherical assemblies
between 8 and 18 nm in diameter with a central cavity (21), which undergo subunit exchange on a timescale of
minutes (22). This exchange is inhibited by
binding to large denatured or partially unfolded proteins, suggesting that subunit
exchange may have an important role in the mechanism of chaperone action (10,22).
αB-Crystallin accumulates in neurons and glia of the central
nervous system under pathological conditions (23), becoming colocalized with αSyn in Lewy
bodies along with a host of other proteins including the sHsp Hsp27, and other
chaperones such as clusterin, Hsp70, and Hsp90 (24). In vitro biophysical studies demonstrated complete
inhibition of the nucleated assembly of αSyn into fibrils
by 0.25 equivalents of αB-crystallin (25). Imaging studies of the aggregation products showed
large amounts of amorphous material, which was interpreted as the binding of
αB-crystallin to monomeric or oligomeric intermediates,
inhibiting the nucleation of fibrillar αSyn aggregates.
Later studies additionally demonstrated that the addition of 0.5 equivalents of
αB-crystallin to aggregating samples of
αSyn is highly effective in inhibiting further growth,
and this was also interpreted as a consequence of the stabilization of monomeric or
prefibrillar αSyn, inhibiting its incorporation into the
growing fibril (26).
Given the complex mechanisms of amyloid formation and the number of aggregated
species involved—e.g., monomers, oligomers, protofibrils, fibrils, and
amorphous assemblies—we were motivated to consider the interaction of
αB-crystallin with additional species along the
aggregation pathway and, in particular, with the mature fibrils themselves, which
hitherto have largely been regarded as inert reaction products. There is evidence,
however, that this view might not be correct, as
αB-crystallin has been reported to bind to
Aβ40 fibrils, and it has been suggested that this
interaction may inhibit further aggregation (13), although the experimental data reported could not conclusively
distinguish between the effects of the chaperone in solution or bound to the fibril.
A separate study of insulin aggregation at low pH demonstrated that fibril
elongation is inhibited after the incubation of the insulin fibrils with
αB-crystallin, where the detection of binding
stimulated the proposal that the latter serves to limit further fibril growth (14). Here we investigate the hypothesis that
αB-crystallin might bind to
αSyn fibrils, and use a variety of techniques including
newly developed QCM and NMR methodologies to explore the range of effects that
result from such chaperone-fibril interactions.
Results
Characterization of <italic>α</italic>Syn interactions with
<italic>α</italic>B-crystallin
The cosedimentation of αB-crystallin and two control
proteins (green fluorescent protein, GFP, and ubiquitin hydrolase, UCH-L3) with
preformed A53T αSyn fibrils was assayed by SDS-PAGE
(Fig. 1
A). A53T αSyn was chosen for this
study because of its high aggregation propensity relative to the WT protein
(4), and it was found to pellet
αB-crystallin in a similar manner to that
previously observed for the WT (25).
The intensity of staining with Coomassie blue was used to estimate the amount of
αB-crystallin present in the pelleted fraction
(Fig. S1
A in the Supporting
Material), and an approximate binding ratio of 0.6:1
αB-crystallin:αSyn was
determined, where the latter refers to the concentration of the constituent
monomers within the fibrils and the observed staining intensity was normalized
by protein mass. The corresponding binding ratios for GFP and UCH-L3 were 0.1:1
and 0.01:1, respectively, both consistent with residual material expected from
the incomplete washing of the pellet, and indicating that the sedimentation of
αB-crystallin was not due to any generic affinity
of proteins for hydrophobic patches on the fibril surface, or rheological
effects from the fibril network during centrifugation.
αB-Crystallin alone was not observed to sediment
under these conditions (Fig. S1,
B and C).
The intrinsic tryptophan fluorescence of αB-crystallin
was used to estimate the concentration of the protein remaining in solution
after incubation with preformed A53T fibrils and centrifugation for 30 min at
16,000 × g (Fig. 1
B); these conditions were found sufficient to pellet
αSyn fibrils completely, and as
αSyn does not contain any tryptophan residues, it
exhibits little intrinsic fluorescence. Only a small (6%) though reproducible
enhancement of αB-crystallin fluorescence was detected
in the presence of monomeric αSyn (Fig. 1
B), with a concomitant blue-shift of 1 nm (343–342 nm)
indicative of the fluorophore being in a more hydrophobic environment,
and consistent with the existence of a weak interaction between
αB-crystallin and monomeric
αSyn (25).
To investigate its putative interaction with monomeric
αSyn further, αB-crystallin
was titrated into uniformly 15N-labeled
αSyn, and changes in the amide chemical shifts were
monitored by recording [1H,15N]-HSQC experiments (Fig. S2). Few significant changes
were observed, and the largest perturbation, at His50, can be
attributed to its high sensitivity to small changes in pH, an observation noted
previously in titrations of monomeric αSyn (8). A small, uniform increase in signal
intensity was observed, in contrast to previous reports of a uniform loss of
intensity (25). This finding suggests
that these changes are more probably attributable to the effect of slight pH
changes on amide exchange broadening than to the association of
αSyn and αB-crystallin.
Thus, whereas measurements of tryptophan fluorescence imply that some
interaction exists between the proteins, the NMR results reported both here and
previously (25) together suggest that
this is only transient and of low affinity, similar perhaps to that
characterized between αB-crystallin and the
amyloid-β peptide (27). For these reasons, these interactions have not
been explored further in this work.
The fluorescence assay was then used to measure the concentration of
αB-crystallin remaining in solution after
precipitation by the addition of αSyn fibrils at two
concentrations. A precise determination of the stoichiometry or affinity of the
interaction between the sHsp and fibrils is complicated by the effect of the
interaction of αSyn monomer with
αB-crystallin, as discussed above, and Rayleigh
scattering resulting from the small number of αSyn
fibrils that remain in solution after centrifugation. In addition, allowance had
to be made for the effect of αB-crystallin on the
monomer-fibril equilibrium (discussed later) and hence on the absolute fibril
concentration. For these reasons, an exact analysis has not been attempted, but
it was observed that binding is proportional to the fibril concentration, and an
approximate ratio of 0.23 ± 0.06 bound
αB-crystallin monomers per
αSyn monomer was calculated. This value is expected to
be a lower bound, with an absolute uncertainty greater than the quoted standard
deviation of the observations, because of the factors discussed above. It is,
however, of a comparable order of magnitude to that estimated by
densitometry (Fig. S1
A).
The binding ratios determined above may be restated in terms of the available
fibril surface area, using a toy model (described in Materials and Methods; see Supporting Material) in which the
αB-crystallin-fibril interaction is approximated as
hard spheres of αB-crystallin monomers packing onto a
cylindrical fibril surface. Although there is evidence that the active subunits
of αB-crystallin may be dimeric (28), provided that both monomers within any such
dimers interact with the fibril surface, we expect that this simple model of
monomeric binding will remain approximately applicable. Such an analysis
determines an approximate maximum binding ratio of 0.90 ± 0.29
αB-crystallin monomers per
αSyn monomer, hence the values determined by
fluorescence and densitometry correspond to surface coverages of approximately
26 ± 11% and 62 ± 20%, respectively. Such high values require
that the chaperone must bind to the overall surface of the
αSyn fibrils, and not just to the small number of
fibril ends.
Inhibition of fibril elongation observed by in situ ThT
fluorescence
Previous studies have examined the effect of
αB-crystallin on the sigmoidal kinetics characteristic
of nucleated polymerization (and fragmentation) reactions associated with the
conversion of αSyn into amyloid fibrils (25,26). Such reactions are
typically highly stochastic, however, and their quantitative interpretation in
terms of individual microscopic processes and rates is complex (7). To simplify the kinetic analysis in this work,
preformed fibrils were used to seed the aggregation reaction in order that the
elongation step alone could be examined. A series of seeded aggregation
experiments were performed in which the initial elongation velocities were
determined as a function of the concentration of
αB-crystallin (Fig. 2
A). These data show clearly that fibril growth was inhibited at
low micromolar concentrations of αB-crystallin. Fitting
to a sigmoidal dose-response curve determined the IC50 (the
concentration of αB-crystallin for half-maximal
inhibition) to be 335 ± 86 nM, although some dependence on the seed
concentration may also be discerned, which shall be discussed later in this
article.
To identify whether or not the inhibition observed above resulted from the
binding of chaperone to αSyn in fibrils or free in
solution, fibrils were preincubated with αB-crystallin
then pelleted, washed, resuspended, and used to seed solutions of monomeric
αSyn. Fig. 2
B plots the resultant kinetic profiles, and shows that
elongation of chaperone-bound fibrils was significantly inhibited
(∼70-fold) relative to untreated fibrils, by analysis of the initial
rates. The residual concentration of αB-crystallin
after pelleting and washing was estimated to be ≤35 nM. This is an
order-of-magnitude below the IC50 and therefore not by itself
sufficient to inhibit elongation, as indicated by the marker in Fig. 2
A. We therefore conclude that the observed inhibition of
elongation results from the specific interaction of
αB-crystallin with the αSyn
seed fibrils.
Inhibition of fibril elongation observed with a quartz crystal
microbalance
To verify the model of inhibition discussed above, an independent technique
utilizing a quartz crystal microbalance (QCM) was employed. QCM is a technique
in which the mass deposited on the surface of a quartz crystal oscillator may be
determined directly via measurement of the frequency of oscillation, and
recently the method has been applied to the study of amyloid growth by measuring
the change in mass of growing fibrils attached to the surface (14). Importantly, this technique enables us to
examine the elongation phase of fibril growth in isolation to other processes
such as nucleation, and this has been used to determine the elongation kinetics
of insulin fibrils grown at low pH, demonstrating the inhibition of their
elongation by αB-crystallin (14). In contrast to insulin fibrils,
αSyn fibrils did not adsorb directly onto the
sensor surface, and instead were covalently attached via lysine side chains, as
described in Materials and Methods (see
Supporting Material). A surface
prepared in this manner is shown in Fig. 3
A, and the fibrils were observed to reproducibly elongate when
incubated with monomeric αSyn (Fig. 3
B). Elongation was readily determined to be proportional to the
monomer concentration (Fig. S3),
as previously observed by solution-state measurements (6), implying that the presence of the surface does
not significantly alter the elongation mechanism.
Fig. 3
C plots the change in deposited mass during a single QCM
experiment. This shows firstly the increase in mass that results from the
elongation of αSyn fibrils in the presence of
αSyn monomer (arrow 1). The
subsequent injection of αB-crystallin with
αSyn monomer (arrow 2) resulted in
a large increase in mass indicative of a binding interaction. After 10 min, the
reaction chamber was washed thoroughly with buffer to eliminate unbound
chaperone, yet upon the injection of fresh αSyn monomer
(arrow 3), further elongation was inhibited approximately
sevenfold. As no free αB-crystallin remained in
solution, inhibition must have resulted from the persistent binding of the
chaperone to fibrils, providing strong support for the solution-state inhibition
results presented previously in Fig. 2.
A second experiment (Fig. 3
D) confirmed this observation: on injection of
αSyn and αB-crystallin, a
large initial increase in mass loading was detected. In contrast to the previous
experiment, this binding was allowed to saturate, and after ∼30 min a
small but constant elongation rate was observed. It is perhaps noteworthy that
this timescale for association is of similar magnitude to that for the subunit
exchange of αB-crystallin (22), and may indicate that the
αB-crystallin subunits are the active chaperone
species. Again, the observed inhibition persisted when the reaction chamber was
flushed with fresh buffer. After overnight incubation of the crystallin-treated
fibrils in fresh buffer, however, the ability of the fibrils to elongate was
partially restored, thus demonstrating the reversibility of the
fibril-crystallin interaction. But the results also imply that dissociation of
the complex occurs only on a timescale of several hours, which is suggestive of
a tight binding interaction.
NMR investigations of the interaction of
<italic>α</italic>B-crystallin with <italic>α</italic>Syn
fibrils
Large species such as amyloid fibrils, with masses of approximately GDa, have
traditionally been assumed to be beyond the reach of solution-state NMR methods.
Recent investigations have demonstrated, however, that in some cases noncore
regions of fibrils have sufficient flexibility to allow us to detect sharp
resonances (29). Therefore, in a
one-dimensional 1H NMR spectrum of
αB-crystallin in the presence of
αSyn fibrils, there are four components that may
potentially be observed: fibrillar αSyn;
residual αSyn monomers; monomeric/oligomeric
αB-crystallin; and fibril-bound
αB-crystallin. Residual monomeric species are key
observables in the study of the thermodynamics of polymerization, and
measurements designed to estimate this concentration have been used to
characterize the energetics of elongation for several fibril-forming systems
(30). The 1H spectrum of
αB-crystallin has previously been characterized,
and reveals that in the native oligomeric complex, the final 12 residues in the
C-terminal extension have sufficient mobility to be observable by solution-state
NMR (18).
Before examination of the fibril-chaperone complex, the spectrum of
αSyn fibrils in solution was explored. Fig. 4
A shows a portion of the one-dimensional 1H NMR
spectrum of a solution of αSyn fibrils (green
line). The fibril spectrum appears almost identical to that of
monomeric αSyn (not shown), albeit with significantly
reduced intensity. Although this spectrum suggests that the bulk of the observed
signal arises from residual monomers in solution rather than the fibrils, a more
detailed study was nevertheless carried out using a series of pulsed-field
gradient (PFG) measurements to define the effective diffusion coefficient
Deff of the species giving rise to the
resonances. For monomeric proteins, Deff
is identical to the translational diffusion coefficient
Dtrans, and its value is independent of the
diffusion delay in the NMR experiment, Δ. By contrast, for very large
molecular assemblies such as amyloid fibrils, Deff
can include contributions from rotational diffusion such that
Deff >
Dtrans for small values of Δ (31). Deff is
shown in Fig. S4 as a function of
Δ for a solution containing αSyn fibrils, and
no such variation was detected. The mean value of
Deff was 8.6(± 1.7) ×
10−11 m2 s−1, which is
entirely consistent with that expected for a small monomeric protein (32). Thus, in contrast to other amyloid
systems such as the SH3 dimer, for which fibril resonances are observable (29), the NMR signal in Fig. 4
A may be attributed to residual αSyn
monomers alone.
Having examined the spectrum of isolated αSyn fibrils
(in equilibrium with residual monomer), it is then possible to analyze their
interaction with αB-crystallin. Fig. 4
A shows the methyl region of the one-dimensional 1H
NMR spectrum of the fibril-crystallin complex alongside spectra of each
component in isolation. Difference spectroscopy, normalized using the internal
DSS reference, reveals that the spectrum of chaperone-bound fibrils is
not the sum of the original components, but that there is additional signal in
the spectrum of the complex. The difference spectrum had, however, the same
appearance as monomeric αSyn (Fig. 4
A). A comparison of intensities in repeated experiments
using independent fibril samples determined that the mean
αSyn intensity in the presence of
αB-crystallin is ∼50% greater than that in
its absence.
To investigate these results further, PFG NMR spectra were recorded to
characterize the diffusion properties of the observed resonances. To limit the
contributions from rotational motion to the diffusion coefficients measured for
fibril-associated species, a long (500-ms) diffusion period Δ was
employed (31). The effective diffusion
coefficients Deff for each sample are plotted in
Fig. 4
B and these data show that the diffusion of
αB-crystallin is retarded in the presence of
αSyn fibrils, consistent with the formation
of a fibril-chaperone complex. The measured diffusion coefficients
correspond, according to the Stokes-Einstein relation, to approximate
hydrodynamic radii of 3.7 ± 0.4 nm and 8.6 ± 0.4 nm for free
αSyn and αB-crystallin,
respectively—in good agreement with previous observations (21,33). The effective diffusion
coefficient of αB-crystallin in the presence of
αSyn fibrils corresponds, when modeled as rotating
rigid rods (31), to a mean fibril
length of 260 ± 140 nm.
The simplest interpretation of these results is that the C-terminal extension of
αB-crystallin is not perturbed by fibril binding
and remains free to tumble in solution; hence, the one-dimensional NMR spectrum
is not altered in the presence of fibrils. However, diffusion is retarded, a
finding consistent with the binding of αB-crystallin to
the fibrils. The concentration of residual αSyn monomer
is also increased in the presence of αB-crystallin,
indicating that the monomer-fibril equilibrium is perturbed by interactions with
the chaperone. Although absolute concentrations of αSyn
monomer were not determined, the observed 50% increase in the monomer
concentration corresponds to a net destabilization of 0.3 ± 0.1 kcal
mol−1 in the free energy of fibril elongation, assuming a
simple polymerization model (30). It is
recognizable that the chaperone-monomer-fibril system comprises many coupled
equilibria, and that there are potentially multiple origins to the observed
increase in monomer concentration. It remains, however, a notable feature of
polymerization reactions that a relatively small change in the net elongation
energy may still result in a large change in the partition of monomers and
fibrils.
Imaging of the fibril-chaperone complex
To confirm the binding of αB-crystallin to
αSyn fibrils in a more direct manner,
immunoelectron microscopy was employed to visualize the fibril-crystallin
complex. αSyn fibrils are of sufficient size that their
twisted ultrastructure could be observed clearly in the electron microscope
after negative staining with uranyl acetate (Fig. 5
A). In contrast, the αB-crystallin
oligomers are more difficult to distinguish, particularly in a multicomponent
system. Samples were therefore incubated with an antibody directed against
αB-crystallin, which was in turn stained with a
secondary antibody conjugated to 10-nm gold nanoparticles. Fig. 5
B shows a sample of
αB-crystallin prepared in this manner and negatively
stained with uranyl acetate; electron dense gold nanoparticles are observed to
be colocalized with the native αB-crystallin
assemblies. The αSyn fibrils shown in Fig. 5
A were also stained with the primary and secondary antibodies
by an identical protocol; no significant nonspecific or background
immunolabeling was observed. An immunolabeled fibril-crystallin complex
containing 0.3 equivalents of αB-crystallin was then
imaged (Fig. 5
C), showing, very clearly, the interaction of
αB-crystallin along the length of the fibrils, with
gold nanoparticles observed on all faces of the fibril surface including
occasional binding at the fibril ends.
Discussion
The results of this article have enabled a novel mechanism to be proposed for the
inhibition of αSyn aggregation by the small heat-shock
protein αB-crystallin.
αB-Crystallin has been demonstrated to bind along the
lateral surface of αSyn amyloid fibrils, and this
interaction has been demonstrated by a range of methods to inhibit elongation
effectively, with half-maximal inhibition at an
αB-crystallin concentration of 335 ± 86 nM.
Although the binding epitopes of αB-crystallin and the
fibrils are unknown, NMR investigations have determined that the C-terminal
extension of αB-crystallin is able to fluctuate freely even
in the bound state, a finding consistent with its purported role as a solubilizing
domain (18). NMR measurements also
indicated that the presence of the chaperone perturbed the monomer-fibril
equilibrium, consistent with a net destabilization of the fibrils by ∼0.3
kcal mol−1. This may indicate that binding of
αB-crystallin along αSyn
fibrils weakens the interactions between adjacent αSyn
molecules, similar to the effect of cofilin binding to actin filaments (34), but the additional effect of a weak
interaction between αB-crystallin and monomeric
αSyn cannot be ruled out at this stage.
In this work, a novel methodology based on QCM measurements has been developed to
monitor αSyn fibril elongation, in isolation from other
processes such as nucleation, and without the need for added probe molecules. As has
been demonstrated in studies of insulin aggregation (14), this technique offers significant promise for the
quantitative investigation of the kinetics and mechanism of
αSyn fibril elongation. To apply this, however, methods
were developed similar to those used in surface plasmon resonance to enable the gold
surface to bind the fibrils. This approach is likely to be applicable to QCM
measurements of other amyloid systems that do not contain accessible thiols or
disulphides.
αB-Crystallin has previously been reported to inhibit
αSyn aggregation, when added even at the midpoint of
sigmoidal assembly curves, and it was hypothesized that the chaperone acts by
stabilizing monomeric or prefibrillar species (25,26). The mechanism put forward in this study, by which
αB-crystallin could inhibit fibril formation by binding
to the fibrils and inhibiting their elongation, suggests that there may be multiple
mechanisms of chaperone action. As binding is likely to occur at least in part to
the hydrophobic clusters that may form in the fibrils, it is likely that
αB-crystallin will also interact strongly with the
oligomeric precursor species, an important property given their cytotoxic nature
(2).
Two mechanisms can be envisaged to rationalize the inhibition of elongation that
results from the interaction of αSyn fibrils with
αB-crystallin: one is a specific capping interaction of
αB-crystallin with the fibril ends, assuming that the
binding here is tighter than that along the length of the fibril; and the second
involves uniform binding of αB-crystallin along the length
of the fibril, where inhibition results from the chance occlusion of the fibril
ends. In this latter binding and occlusion model, inhibition is expected to be
observed only in the presence of near equimolar ratios of
αB-crystallin and αSyn fibrils,
and the dependence of the IC50 upon the seed concentration noted in Fig. 2
A would be consistent with such a mechanism. Further investigations
of the stoichiometry and affinity of the fibril-chaperone interaction will, however,
be required to differentiate these models with confidence.
A key question in the study of sHsps is whether the native oligomers act as
chaperones directly, or whether dissociation into subunits occurs before their
binding (35). The approximate binding
ratios presented in this work would be consistent with either a relatively uniform
binding of monomeric or dimeric αB-crystallin subunits
along the fibril surfaces, or a rarer binding of the oligomers. The high
surface coverage that would result from the subunit binding appears attractive as a
means of explaining the efficacy of inhibition, particularly as this effect persists
when the chaperone is removed from the solution after binding has occurred. Although
this mechanism of inhibition has not been put forward previously, it is consistent
with previous studies in which sHsp subunit exchange dynamics have been correlated
to chaperone activity, in both αB-crystallin and its
phosphorylated derivatives (22,36).
These findings present the suggestion that other sHsps may act by a similar
mechanism. The Hsp20 from the bovine parasite Babesia bovis
inhibits Aβ40 amyloid formation, and the chaperone
was active only at low (10–100 nM) concentrations in which dimeric subunits
were prevalent, whereas at higher chaperone concentrations, in which larger
oligomers became populated, the inhibition of aggregation was no longer observed
(37). Additionally,
αB-crystallin may bind to components of the
cytoskeleton including intermediate filaments and microtubules, although electron
microscopy has demonstrated that such binding occurs via the oligomeric complex
rather than isolated subunits (38). This
suggests that different chaperone activities (e.g., suppressing amyloid formation,
thermally induced amorphous aggregation, and cytoskeletal binding) correspond
to a variety of phosphorylation or oligomerization states and binding modes.
For example, lysozyme unfolding intermediates are bound by multimeric Hsp27 (39), and whereas
αB-crystallin activity against
αSyn amyloid formation increases with temperature
(correlating with increased subunit exchange), little variation with temperature was
observed against the thermally induced amorphous aggregation of alcohol
dehydrogenase and citrate synthase (40).
Phosphorylation-mimicking mutations have also been demonstrated to differentially
regulate the chaperone activity of αB-crystallin against
amyloid fibril formation and amorphous aggregation (36). Equally, however, some of this variation may result
from a degree of specificity of individual members of the sHsp family (41).
αB-Crystallin has shown no evidence of ATPase or refolding
activity (19). Instead, sHsps have been
proposed to bind to misfolded states before interaction with other chaperone
systems, which may facilitate refolding or degradation (41). Horwitz et al. (9) has suggested that
αB-crystallin in the eye lens may act as an irreversible
sink for unfolded or misfolded proteins, as there is no evidence of further
chaperone systems within the lens, and active protein synthesis does not occur once
the lens proteins are laid down. The binding of
αB-crystallin to αSyn fibrils may
therefore be a protective mechanism to stem the growth of amyloid deposits
before clearance by further quality control mechanisms within the cell (41). This hypothesis is supported
by a mouse model of PD expressing A53T αSyn, in
which selective upregulation of αB-crystallin was observed
in affected glial cells (42). Additionally,
we suggest that binding of αB-crystallin to fibrils may
reduce their cytotoxicity by camouflaging exposed hydrophobic surfaces. This
hypothesis is supported by a tissue culture model of Lewy body disease in which
colocalization of αB-crystallin with
αSyn deposits occurs without a reduction in the number
of inclusions compared to cells not transfected with
αB-crystallin, although there is a significant reduction in
cytotoxicity (43). Finally, as exposed
fibril surfaces have been proposed to act as sites of secondary fibril nucleation
(44), the coating of
αSyn fibrils by αB-crystallin
could inhibit not just their elongation but further nucleation processes as well. It
is also possible, however, that fibril binding by chaperones may have a deleterious
role by sequestering them and reducing their efficacy in other physiological
functions, e.g., interacting with misfolded monomers or oligomers, or, indeed,
helping in regulation of the cytoskeleton (41). Certainly, these findings reveal novel activities of the cellular
proteostasis network, which is of growing interest in the field of systems
biology, and an increasing understanding of its behavior may contribute to
the rational development of therapeutic proteostasis regulators (45).
This finding is also likely to have general relevance to other amyloid and chaperone
systems. Indeed, recent work suggests that another chaperone, Hsp104, is capable of
suppressing amyloid growth at many stages along the aggregation pathway, including
the elongation of mature fibrils (46).
Fibril binding and inhibition of elongation by
αB-crystallin has previously been demonstrated for the
Aβ40 peptide, although no causal link was demonstrated
(13). Inhibition of
β2-microglobulin elongation by
αB-crystallin at low pH was also reported (13), and inhibition of insulin
aggregation under similar conditions has also been observed (14). This work is therefore important in
extending mechanistic studies of αB-crystallin to
physiological conditions and, given apparent similarities in the behavior of
αSyn, Aβ40,
β2-microglobulin, and insulin, suggests a
generic mechanism of αB-crystallin action.