Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these “neurotoxic proteins” triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases.
Amyloid-related neurodegenerative diseases are incurable disorders that are currently classified as conformational diseases (a subset of the proteinopathies). These diseases are thought to be primarily caused by an unknown conformational change in specific proteins, termed “neurotoxic proteins” (NPs), which confers them a cytotoxic gain of function. This initial transition in the soluble monomer triggers a series of events that lead to the formation of small soluble oligomers and protofibrils, and ultimately the generation of highly ordered fibrillar insoluble aggregates. More than 20 of these NPs have been found to be intrinsically disordered proteins (IDPs). Despite the fact that the monomers of NPs have different primary and, when folded, tertiary structures, their oligomers and aggregates share common structural properties [1], [2], including recognition by a conformational antibody [3]. Whereas the nature of the cytotoxic species and the molecular mechanisms involved in their misfolding and aggregation remain largely unknown, there is growing evidence that conformational changes preceding aggregation are fundamental in the molecular pathogenesis of these diseases [1]. Furthermore, as the formation of monomeric misfolding intermediates is regarded as the earliest event in the pathogenic cascade, this process represents an ideal therapeutic target [4]. Remarkably, it was recently demonstrated that the key conformational change that endows amyloidogenesis occurs in the monomer, triggering aggregation and fibrillogenesis as well as cytotoxicity [5]–[7]. Thus, to understand the acquisition of toxic properties by NPs, the three-dimensional structure of this toxic monomeric conformer must be characterized. However, the propensity of monomers to aggregate in vitro hampers their structural analysis using classical high resolution techniques such as X-ray crystallography or solution nuclear magnetic resonance (NMR). Nevertheless, it was shown that some NPs are rich in α-helical [8], [9] or disordered [10], [11] segments. It must be noted that the oligomeric state of α-synuclein, the NP associated to Parkinson disease, remains controversial. Thus, while some reports validate it as a natively unfolded monomer [12], other studies have shown, interestingly, that its potential native state is a dynamic α-helical tetramer [13], [14] that undergoes unfolding/folding cycles associated to its physiological function to promote SNARE complex formation in neurons [15]. Furthermore, it was suggested that many alternative monomeric conformations may coexist in a dynamic equilibrium that progressively shifts toward conformers enriched in β-structure, which are thought to be the first toxic species [2]. However, the inherent ensemble averaging of these techniques prevents the full complexity of monomer conformational equilibria from being elucidated and the fast fluctuations involved from being resolved. Moreover, solid-state NMR and X-ray crystallography can only provide structural information about the final species (i. e. , amyloid fibers [16]). Bulk-based, theoretical, and single-molecule studies support the existence of conformational heterogeneity in intrinsically disordered NPs [1], [17], [18]. However, the co-existence of several species in solution at equilibrium, some of which may be scarcely populated, makes analyzing IDPs a particularly challenging task using established biophysical techniques [19]. By contrast, single-molecule techniques provide a unique opportunity to tackle this problem being able to reveal monomeric conformers [18]. Some of these techniques have already been used to monitor the activity and conformation of proteins with conformational plasticity [20], resolving fast fluctuations (20–300 ns) at the monomeric level [21]. Of the available techniques, nanomanipulation has two additional advantages. First, it allows the forces involved in stabilization of the intramolecular interactions to be directly measured (mechanical stability); and second, it enables the mechanical stability of a protein to be related with its conformation. In particular, atomic force microscopy (AFM) -based single-molecule force spectroscopy (SMFS, for short) shows remarkable potential to analyze the conformational equilibrium of monomers, and even to detect low-abundant conformers [18]. In principle, measuring the mechanostability of NPs using SMFS should be feasible, as the toxic gain of function of NPs parallels the acquisition of β-structure [1], [2], a secondary structure that is usually detectable and is often more mechanostable (M) than α-helical or disordered (random coil [RC]) conformations [22]. Furthermore, determining the mechanostability of NPs is particularly relevant, given their processing by chaperones and chaperone-related proteases [23], which unfold their substrates mechanically using AAA+ ATPase molecular motors [24], [25]. Previous SMFS studies of NPs [26]–[29] have been hindered by three serious problems. Firstly, standard polyproteins were used as single-molecule markers fused to the NP, and since the NP is placed in series with the marker repeats, the SMFS recordings obtained are typically contaminated in the proximal region (Figure 1), thereby compromising the reliability of the data obtained. Secondly, the amyloidogenic behavior of the NP fused to the polyprotein was not demonstrated. Finally, there is no convincing evidence demonstrating that the selected data originated from bona fide intramolecular interactions (rather than intermolecular interactions). In the present study, these limitations have been overcome to reveal the existence of a rich conformational polymorphism in the monomer of NPs and to demonstrate its link to amyloidogenesis and neurotoxicity (Figure S1).
To circumvent the first of the aforementioned limitations, we recently developed a novel strategy that uses a new vector to mechanically protect the NP as a guest inside a carrier protein of well-known mechanical properties (plasmid for force spectroscopy-2 [pFS-2]) (Figure 1A) [30]. Accordingly, unlike previous works [26], by means of this approach (termed the “carrier-guest” strategy) the SMFS data obtained from the NP are always read after the unfolding of the carrier, far from the proximal region to the AFM substrate, which is known to be typically noisy and thus unreliable (Figure 1B; Text S1). We used SMFS to analyze the nanomechanics of NPs expressed as fusion polyproteins in pFS-2. Using the so-called length-clamp mode of this technique, two basic parameters are directly obtained (see Text S1): the mechanical stability of the resistance barriers (F, measured directly by the height of the force peaks in a force-extension recording), and the length released after unfolding (increase in contour length (ΔLc) as measured by fitting the force-extension recording to the worm-like chain [WLC] model of polymer elasticity). We followed highly stringent criteria when selecting single-molecule recordings using the carrier-guest mechanical protection strategy whereby we only selected those recordings in which the ΔLc of the carrier (alone or including a non-mechanostable fraction from the NP) was followed, although not necessarily consecutively [29], by force peaks with ΔLc values that accounted for the total expected length released by the carrier-guest protein (Figure 1B; Text S1). This strategy was employed to study four model NPs: three human NPs (expanded poly-glutamines -polyQ-, β-amyloid1–42 -Aβ42-, and α-synuclein) and a neurotoxic-like protein that has been extensively used as a model for human prions (Sup35NM, a yeast prion). These NPs are amyloidogenic IDPs [18], and they are thought to cause the most representative neurodegenerative diseases: polyQ (familial disorders such as Huntington' s disease), Alzheimer' s, Parkinson' s, and prion diseases, respectively [1], [18]. All these NPs, including Aβ [31], are found intracellularly, and they are therefore susceptible to be stretched by the protein-processing machinery [23]. To test whether the properties of these engineered NPs and carriers are preserved in the carrier-guest proteins (the second limitation of the former SMFS studies listed above), we performed standard structural, calorimetric, and fibrillogenic experiments on the isolated carrier-guest proteins (Figures S2, S3, S4, S5, S6, S7, S8, S9; Table S1; Text S1). Whereas the conformational stability of the carrier proteins is reduced when they contain the guest NP, and in some preparations some molecules even lose their folded tertiary structure, the carriers essentially maintain their structure and mechanical properties (Table S2), which enables the unambiguous observation of the conformational polymorphism of the monomeric NPs. Furthermore, regarding the possible presence of spurious interactions, in these studies we found no evidence of contacts or other interactions between the carrier and guest proteins beyond the covalent linkage of the polypeptide chain. This rules out the possibility of artifactual contacts, and hence a possible gain of structure, induced in the NPs when nested inside the carriers or the other way around. To the best of our knowledge, the pFS-2 vector is the first strategy capable of successfully avoiding the contamination of SMFS data with non-specific interactions from the proximal region of the AFM, which is particularly useful for studying IDPs [30]. Indeed, this approach guarantees that the selected data originate from the stretching of single NP molecules (Figure 1). However, in principle, the stretching of a single NP monomer does not exclude possible interactions of the NP with the carrier, the AFM elements (substrate or tip) or other surrounding NPs (intermolecular interactions from oligomers and/or fibers). Such potential interactions (the third limitation of those SMFS studies listed above) must be ruled out before force events can be unequivocally attributed to intramolecular interactions in the NP monomers. By using specific controls, we provide compelling evidence that in our experimental conditions, the stringently selected SMFS data (we sampled about 100 molecules of each NP with each set of recordings resulting from 50,000–100,000 pulling attempts) exclusively arose from monomeric NPs and, therefore, represent bona fide intramolecular interactions (Text S1). Finally, by restricting the degrees of freedom of the hosted NPs, the pFS-2 strategy has the additional advantage of dramatically slowing down fibrillogenesis (Figure S6D; Text S1), thereby minimizing the formation of aggregates, during the nanomechanical analysis of the monomeric NPs. PolyQ diseases are unique among amyloidogenic neurodegenerative diseases in that they are both genetically determined and their NPs, polyQ expansions, have pathological thresholds (21–30 and 36–40 glutamine residues in spinocerebellar ataxia type 6 [SCA6] and Huntington disease, respectively). Interestingly, there is a positive correlation between the number of glutamine repeats and the severity of the disease, the age of onset, and the oligomerization kinetics [4], [32]. Thus, in the simplest hypothesis, the disease determinants are expected to correlate with the number of glutamine residues. Using SMFS we looked for possible mechanical differences in the monomeric polyQ tracts of three lengths that are considered to be sub-, near- (except for SCA6) and super-threshold for triggering polyQ diseases: Q19, Q35, and Q62 (Figure 2A). We found that the subthreshold tract showed no force peaks; we term these conformers “non-mechanostable” (NM) (i. e. , there were no detectable SMFS conformations; the force detection limit of SMFS was taken as twice the force error, 20 pN, at a pulling speed of 0. 4 nm/ms) (Figure 2A, orange bars; this representation is followed also in Figures 3–5; Text S1). Similar mechanical behavior was observed for non-amyloidogenic/non-neurotoxic IDPs [33], [34], including VAMP2 (Figure 2B) [35]. Accordingly, neither Q19 nor VAMP2 constructs produced amyloidogenesis (Figures 2, S2C, S2D, S3C, and S3D). By contrast, Q35, which showed moderate amyloidogenesis (Figure S2C and S2D), exhibited not only numerous NM conformations (F≤20 pN) but also several force peaks with a variety of ΔLc values; we call these conformers “mechanostable” (M) (F>20 pN) (Figure 2A, red bars). ΔLc and F were found to be uncorrelated (Figure S10A) as recently reported for the NP tau [29]. Interestingly, this incipient conformational polymorphism included conformers with extremely high mechanical stability (F≥400 pN). Given that the mechanical stability (upon N-C stretching) of eukaryotic proteins studied to date is below 400 pN [22], we have operationally defined an additional subset in the latter class based on an arbitrary force value of 400 pN. This subset of M conformers that accounts for events with F≥400 pN (which are rare) is termed hyper-mechanostable (hM). Based on our current knowledge of NPs [1] and recent studies of polyQ tracts [5], it is likely that NM and M conformers correspond to RC and β-stranded structures, respectively (see Text S1), as has been previously assumed [26], [27]. Together, these results indicate that polyQ tracts of 35 glutamines can experience a structural transition that permits conformational fluctuations. Significantly, Q62 exhibited even greater conformational polymorphism that also included a higher number of hM conformers (Figure 2A). Thus, expanded polyQ tracts (Q35 and Q62) allow the appearance of conformations that can be detected by SMFS; these mechanical conformations include hM conformers. This polymorphism (estimated as the frequency of M conformers, including hM ones) is positively correlated with the polyQ length, which in turn correlates with amyloidogenesis. Interestingly, a structural correlation with the pathological threshold for polyQs has been previously reported [5]. The SMFS analysis of the three NPs used as models of sporadic neurodegenerative diseases (Aβ42, α-synuclein, and Sup35NM) [1], [2] showed that the wild-type (wt) form of each protein exhibited a rich conformational polymorphism, which also included rare hM conformers (except for Aβ42) (Figures 3–5). The differences in the occurrences of stable conformers in NPs relative to non-amyloidogenic IDPs (VAMP2, Q19, and F19S/L34P Aβ42, see below) are statistically significant (Table 1). In the wt form of the four model NPs, the frequency of M conformers ranged from 0% in Q19 (Q21 is the minimum pathological threshold detected in polyQ diseases, specifically in SCA6 [2]) to 64% in Sup35NM, which for each NP was positively correlated with amyloid fiber density (Figures 2–5, S2D, S3D, S4D, S5D, S6D; Table S1). The frequency of hM conformers for wt NPs ranged from 0% to 8% (Figures 2–5). These rare events are associated with extremely high rupture forces (up to ∼800 pN), greater than those reported for the most mechanostable proteins described to date (i. e. , bacterial cohesin modules [36]) and just below the force range required to break a covalent bond [37]. Table 1 summarizes the frequency for each conformer type. Frequently, more than one force peak was found for each NP molecule (Figure S11), suggesting the presence of more than one structured region in the M conformers. It should be noted that the number (and thus the frequency) of M conformers may in reality be greater than that estimated due to the existence of additional SMFS curves that could correspond to incomplete recordings, i. e. , compatible with the unfolding of a carrier bearing a mechanostable element that was not fully observed probably due to the premature detachment of the molecule. Although mechanostability is a stochastic property, these premature detachment events should often be lower than the forces needed to unravel the undetected NP (putative M conformers). A subset of these recordings contained putative hM events (Figure S12). Although these putative events were not included in the sample size, n, it is interesting to note that there is a good correlation between the putative and detected hM events among the different NPs and conditions tested (Table S3). Taken together, SMFS analysis revealed that whereas VAMP2, a non-neurotoxic IDP, always shows a low (undetectable) resistance to mechanical unfolding, all the NPs studied behaved similarly, displaying a high degree of mechanical conformational polymorphism that often included a few hM conformers. We postulate that this behavior is associated with amyloidogenesis and neurotoxicity, which, in agreement with previous results [5], strongly suggests that a critical conformational change in the monomer leads to the generation of the first toxic NP species. Hence, we have tested this working hypothesis by studying four conditions in which the disease and/or amyloidogenesis are affected in two ways: diminished with a peptide that blocks the oligomerization process, an aggregation-defective mutant, or a peptide that inhibits the β-conformational change; and intensified by familial-disease mutations. According to our hypothesis, in these conditions the conformational polymorphism (and hM frequency) should be unaltered, abolished, decreased, or increased, respectively. We first studied the effect of blocking oligomerization on the conformational polymorphism of monomeric NPs. Specifically, we used the SV111 peptide, which inhibits Aβ42 oligomerization and fibrillogenesis by binding to a specific β-hairpin structure [38], but should not affect the conformational fluctuations of the unbound monomers. Notably, incubation of Aβ42 with SV111 (100 µM [38]) yielded SMFS results similar to those from the untreated Aβ42, including the number of events per molecule (Figures 3 and S11; Table 1), whereas fibrillogenesis was inhibited (Figures 3 and S4D; Table S1). Since this peptide effectively inhibits oligomerization, this experiment also served as a control to rule out a significant contribution of intermolecular interactions to our SMFS data, strongly suggesting that the behavior observed originated from bona fide intramolecular interactions within the NP monomer (Text S1). We then analyzed a double mutant of Aβ42 known to suppress fibrillogenesis (F19S/L34P) [39]. As expected, the results obtained (Figures 3 and S4) were similar to those obtained with Q19 or VAMP2 (Figures 2, S2, and S3). This experiment also served as a control to exclude potential interactions between the NPs and the AFM elements as a source of the M events (Text S1). We next studied the effect of additional representative familial neurodegenerative disease mutations on the conformational polymorphism, using dominant forms responsible for the early onset of the disease and for the acceleration of oligomerization kinetics [1], [2]. In addition to Q35 and Q62, we also studied the arctic (Arc: E22G) mutation in Aβ42 that has been implicated in familial Alzheimer' s disease, as well as the A30P and A53T mutations in α-synuclein that are involved in familial Parkinsonism [1], [2]. Like the results obtained with Q35 and Q62, we found an increase in the frequency of M conformers, including hM ones, for all the mutants analyzed (Figures 3 and 4; Table 1). Similarly, the number of mechanostable regions (i. e. , force peaks) per molecule increased for each NP mutant studied (Figure S11). Finally, two of the familial-disease mutations also increased the number of putative hM recordings (Q62 in polyQ tracts and A53T in α-synuclein) (Figure S12). Taken together, these results indicate that familial-disease mutations increase the propensity to form M conformers, including hM forms, an effect that is correlated with aggregation and fibrillogenesis, at least for polyQ tracts. This increased conformational polymorphism may also explain the dominant character of these NP mutations. We finally analyzed how the conformational polymorphism of NPs was affected by PolyQ-binding peptide 1 (QBP1), an amyloidosis inhibitor known to block the critical β-conformational change of expanded polyQs at the monomer level. QBP1 binds RC conformers [5], [40], suppressing oligomerization, amyloidogenesis, cytotoxicity [5], and neurodegeneration [41]. Specifically, we used the minimal active core of this peptide (QBP1-M8) [42]. Incubation of Q62 with QBP1 completely abolished the formation of fibers (Figures 2 and S2; Table S1), and effectively reduced both the frequency of detected and putative M conformer formation, including hM forms (Figures 2, S12B; Tables 1 and S3). The number of mechanostable events per molecule (Figure S11A) was also reduced. In what was originally intended as a negative control experiment, we also tested the effect of this peptide on the other three NPs, selecting two well-characterized familial-disease mutations (Arc Aβ42 [Figure 3] and A53T α-synuclein [Figure 4]) and the yeast prion Sup35NM (Figure 5). Surprisingly, QBP1 strongly diminished the formation of M conformers (detected and putative) of both A53T α-synuclein and Sup35NM (Figures 4,5, and S12; Table S3). The subset of hM conformers (detected and putative) showed a significant decrease as well (Figure S12; Table S3). Aggregation and fibrillization were also reduced or suppressed by QBP1 in these two NPs (Table S1). It should be noted that the effect of the peptide on Sup35NM was incomplete as some fibers were still formed (Figures 5 and S6D), while its effect on A53T α-synuclein was not as significant, given the small number of fibers formed by this mutant in the absence of the inhibitor (Figures 4 and S5D). Interestingly, in the specific conditions used in our experiments, QBP1 appeared to have no significant effect on Arc Aβ42, either on the conformational polymorphism or its aggregation and fibrillization capabilities (Figures 3, S4C, and S4D; Tables 1 and S1). Furthermore, the frequency of hM events in the untreated Arc Aβ42 was so low (the lowest of the four NPs) that any apparent effect could not be considered significant. To rule out any effect of QBP1 on Aβ42, higher concentrations of QBP1 (from 100 µM to 1 mM) were tested on the isolated Aβ42 and no effect in fiber formation was observed (Figure S4D). These results point to the existence of common characteristics at the monomer level in at least three of the NPs studied (expanded polyQs, α-synuclein, and Sup35NM), which appear to be recognized by QBP1. In spite of the similarities found for all the NPs studied, the inability of QBP1 to recognize Aβ42 indicates that there may be some variation in nascent amyloidogenesis. It should also be noted that structural characterization as well as aggregation and fibrillogenesis experiments (Figures S2, S5, and S6) suggest that the inhibitory effect of QBP1 is stronger on expanded polyQ tracts than on A53T α-synuclein or Sup35NM, while it seems not to affect Arc Aβ42 (at the same peptide concentration) (Figure S4), in accordance to its mentioned effect on the conformational polymorphism. Given that QBP1 blocks the formation of β-structures [5], it is likely that at least some M conformers contain such β-structures, as discussed below. These results extend previous observations of common molecular mechanisms in both oligomerization and fibrillogenesis on the basis of the recognition of similar oligomeric structures in all NPs tested by the A11 conformational antibody [3] and in the existence of a similar though somewhat variable cross-β spine structure discovered in these fibers [16], [43]. Thus, our results point to the existence of additional common molecular mechanisms upstream in amyloidogenesis acting in the initial stages of this process at the monomer level.
Single-molecule techniques are ideal to analyze the conformational polymorphism of NPs and SMFS in particular is a highly promising technique that has already been used to this end [26]–[29]. However, the disordered nature of NPs and their tendency to oligomerize pose significant challenges to this kind of analysis. We have successfully developed a strategy to unequivocally analyze at the single-molecule level the conformational polymorphism of IDPs, and in particular NPs. Accordingly, we have sampled the conformational space with about 100 molecules from each of the four most representative NPs and their variants: polyQ tracts, Aβ42, α-synuclein and Sup35NM (see Table 1). We demonstrate that proteins (neurotoxic or not) that do not form amyloid fibers (i. e. , non-pathological polyQ tracts such as Q19, VAMP2, and the F19S/L34P Aβ42 mutant) show no mechanical conformational polymorphism, as monitored by SMFS. By contrast, proteins with propensity to form amyloid fibers (expanded polyQs, Sup35NM, and wt and familial-disease mutations for Aβ42 and α-synuclein) exhibit a rich conformational polymorphism that includes rare hM conformers. As mentioned, it is likely that NM and M conformers correspond to RC and β-stranded structures, respectively. In the case of prions, conformers separated by a high-energy barrier and stabilized by strong non-covalent forces (presumably high-density hydrogen bonding), have recently been proposed to account for the heritability of conformations [44]. It is tempting to speculate that these structures may contain β-strands in a shear (rather than zipper) configuration, as this is the most mechanostable structure observed experimentally to date [36], [45]. These hM conformers were already suggested in previous SMFS studies of polyQs [28], although the authors assumed they involved collapsed rather than β-stranded structures, even for non-pathological tracts. It is important to emphasize that our SMFS results correlate closely with our structural and aggregation/fibrillogenesis controls (Table S1). However, our results are not directly comparable to those of previous studies, which did not include unequivocal single-molecule markers, structural controls, or controls for intermolecular interactions. Thus, while our findings are in line with previous results from tau [29], except that these authors did not report NM conformers, they diverge significantly from those reported for α-synuclein [26], [27], possibly owing to the larger sample size used for tau as compared with α-synuclein (214–453 versus 34–63, respectively). Neurotoxic and neurotoxic-like proteins involved in sporadic neurodegenerative diseases (Aβ42, α-synuclein, and Sup35NM) [1], [2] exist as a broad ensemble of NM and M conformers, including a few hM forms. In contrast, Q19 (which fails to trigger disease) does not show M conformers. These findings and previous observations [41] strengthen the connection between M conformers, amyloidogenesis, and disease. Indeed, mutations that induce an early disease onset and accelerate amyloidogenesis [1], [2] increase the frequency of M (and hM) conformers. Conversely, treatment with the QBP1 inhibitor peptide strongly reduced the frequency of M (and hM) conformers in A53T α-synuclein and Sup35NM. Intriguingly, this treatment did not affect the conformational polymorphism of Arc Aβ42. Moreover, incubation of Aβ42 with SV111 yielded a similar conformational polymorphism as for the untreated Aβ42, although it did inhibit aggregation and fibrillogenesis, in agreement with previous results [38]. Overall, we observed an excellent correlation between the propensity to develop a neurodegenerative disease, previously demonstrated by others [1], [2], and conformational polymorphism, including hM conformers, of the relevant NPs. We show that this polymorphism can be altered in both directions, towards more “structured” conformers by pathological mutations or towards the NM conformation, by the QBP1 inhibitor and by non-fibrillogenic mutations. The dramatic reduction of the frequency of M conformers, including hM forms, produced by QBP1 suggests that hM conformers and/or their precursors from the M pool are good candidates to represent the primary cause of neurodegenerative diseases (i. e. , the hypothetical initial misfolded intermediate). Since QBP1 inhibits the formation of these conformers and exerts anti-neurodegenerative effect [41], we conclude that hM conformers, and/or their precursors, are likely on-pathway intermediates in amyloidogenesis and/or cytotoxicity. The discovery of these common features offers hope that a single therapeutic agent may be able to prevent, halt, or reverse the development of many neurodegenerative diseases arising from these different proteins. Prior to our work, it was known that common molecular mechanisms existed in the later stages of amyloidogenesis, including oligomerization [3] and amyloid fiber formation [43]. Also, prion-like behavior has been recently shown for polyQ tracts, Aβ and α-synuclein [46]. Our identification of a common pattern of conformational propensities among the monomers of the four most representative NPs further extends this unifying view to the earliest stages of the amyloidogenesis process. Furthermore, the inhibition of fibrillogenesis by the same peptide in three out of those four NPs suggest that amyloidogenesis shows common molecular features from the very beginning of the process, although some diversity is also likely [16]. In general, our results strongly support the conformational change hypothesis as the mechanism to explain the initiation of amyloidogenesis [47], wherein amyloidogenesis is thought to be preceded and triggered by a conformational change in the monomer precursor [4]. In the particular case of polyQs (the clearest-cut system), the nature of the conformational changes that occur during polyQ amyloidogenesis have remained controversial [2], [4], [48]. In fact, other models for polyQ diseases have received greater attention in the literature, such as the “polymerization hypothesis” (i. e. , the conformational change is induced upon monomer polymerization into fibers) or recruitment mechanisms like the “linear lattice” model [2], [48]. Our data strongly suggest that it is the monomer that undergoes the critical conformational change that results in the generation of the first toxic species [5]. Furthermore, our data provide strong evidence supporting the so-called “structural threshold hypothesis, ” which states that above the pathological threshold polyQ tracts undergo a structural transition from RC to β-sheet structure [49] and indicating that it occurs in the monomer. Our single-molecule approach also provides an explanation for the failure to detect this change using NMR spectroscopy in a previous study [50]. M conformers represent just 7. 5% or less of species, a fraction that is difficult to detect even if all these conformers were from a unique species (which seems not to be the case according to our ΔLc data). Finally, we note that in the aforementioned study the Glutathione S-transferase-Qn (GST-Qn) fusion proteins used did not show evidence of aggregation. We have adopted a new strategy to unequivocally identify and quantify the conformational polymorphism of NPs and their pathological and non-pathological variants. This approach permits—for the first time to our knowledge—rare hM conformers to be unequivocally detected. We postulate that these conformers, or their precursors, may mediate the primary event in the pathological cascade leading to amyloid fiber formation and concomitant neurodegenerative disease. On the basis of these findings, we propose the self-explanatory term “proteinoscleroses” to collectively refer to amyloid-related neurodegenerative diseases. In this context, hM conformers (or their earliest precursors, if we assume a sequential pathway) constitute ideal pharmacological targets and biomarkers of the propensity to develop such diseases. Moreover, the conformational inhibitor QBP1 appears to target the pathway that leads to hM conformer formation in expanded polyQs, α-synuclein, and Sup35NM prion. Whereas there is evidence for rapid interconversion of conformers in NPs [21], we propose that once formed, the hM conformers are likely to be kinetically trapped (because of the high energy unfolding barriers) and might mechanically jam the unfoldases of the protein-processing machinery of the cell as postulated previously [51]. Such an effect would slow down their processing, which would unbalance the cell proteostasis by increasing the cytosolic concentration of hM conformers. This imbalance could affect distinct cellular processes and ultimately, it may lead to the formation of oligomers and amyloid fibers (Figure S1). There are three findings that support this hypothesis: (1) amyloid-related neurodegenerative diseases have a late onset, developing as aging impairs the efficiency of the protein-processing machinery [52], [53]; (2) the proteasome is transiently impaired in vivo by the expression of the N-terminus of pathologic huntingtin [54]; and (3) the AAA+ ATPases from the degradative machines have recently been shown to unfold their substrates mechanically using relatively low forces (with a pulling geometry apparently similar to that of SMFS experiments and a low calculated loading rate of 0. 15 pN/s) [24], [25]. Alternatively, toxic NP conformers may sequester other IDPs involved in essential cell functions [55] or affect the normal lysosomal autophagy process [56]. It should also be noted that the causal relationships between misfolding and oxidative stress remain a matter of debate [57]. In spite of this, it must be noted that, alternatively, hM conformers may represent dead-end species, i. e. , by-products, off-pathway of amyloidogenesis and cytotoxicity; in this case the toxic species would be part of the remaining species of the M pool. In conclusion, our findings show a clear correlation in all NPs studied between conformational polymorphism (including the presence of hM conformers) and neurotoxicity. They strongly indicate that hM events represent highly collapsed or structured conformers in the fibrillization pathway and that these conformers, or their precursors, may be the primary cause of amyloid-related neurodegenerative diseases. Our approach opens the door to understanding the molecular mechanism of amyloidogenesis and developing a single therapeutic strategy that may treat distinct neurodegenerative diseases using a single drug. Our novel approach also offers a new means of studying other proteins involved in conformational diseases and should allow the dissection (by using the appropriate conditions) of the toxicity and fibrillogenic pathways involved in amyloidogenesis.
Polyprotein engineering was performed using a new vector recently described by our group (pFS-2) (Table S4) [30]. Detailed methods on polyprotein sample preparation, AFM-SMFS, circular dichroism (CD), differential scanning calorimetry (DSC), NMR, turbidimetry, Congo Red binding assay, transmission electron microscopy (TEM), and imaging AFM experiments can be found in Text S1.
Neurodegenerative diseases like Alzheimer' s or Parkinson' s are currently incurable. They are caused by different proteins that, under certain circumstances, aggregate and become toxic as we grow older, but the molecular events underlying this process remain unclear. The lack of a well-defined structure, and the tendency of these “neurotoxic proteins” to aggregate make them difficult to study using conventional techniques. Here, we use an established single-molecule manipulation technique combined with a new protein-engineering strategy to show that all these proteins can adopt a rich collection of structures (conformers) that includes a high proportion of mechanostable conformers, which are associated with toxicity and disease. We also find that a known drug can block the formation of these mechanostable structures in different neurotoxic proteins. We suggest that the most mechanostable conformers, or their precursors, may trigger the pathogenic cascade that results in toxicity. We thus propose that these mechanostable structures are ideal targets for early diagnosis, prevention, and treatment of these fatal diseases.
medicine
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drugs and devices
chemical biology
biomechanics
protein folding
protein structure
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chemistry
biology
recombinant proteins
biophysics
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biochemistry
protein chemistry
Common Features at the Start of the Neurodegeneration Cascade