Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis

Nanomechanical analysis of NPs using the pFS-2 strategy.

<p>(A) Top: Schematic representation of the pFS-2 polyprotein <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Oroz1" target="_blank">[30]</a> carrying a guest NP (in orange) mechanically protected within a carrier module (C, in gray), flanked by ubiquitin repeats (U, in black). Bottom: cartoon representations of the two carrier-guest constructions used in this study: ubiquitin (left, PDB code 1d3z) and titin I27 (right, PDB code 1tit). The images were prepared by VMD 1.8.6 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Humphrey1" target="_blank">[58]</a>. The hydrogen bonds of the mechanical clamp in both carriers are indicated by black bars <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-CarrinVzquez2" target="_blank">[51]</a>. (B) Representative force-extension recordings of pFS-2+Sup35NM. Using this strategy, we can unambiguously resolve a variety of conformations adopted by Sup35NM, ranging from a typical NM conformation (top trace, in orange), to M conformations with different degrees of mechanical stability (in red), including hM conformers (bottom). This color code will be maintained throughout the rest of the article. On the basis of our carrier-guest design, the carrier module must always unfold completely (“a” is Δ<i>L</i>_c for the carrier module) before the force can access and stretch the guest NP, resulting in its unfolding (“b” and “c” represent the Δ<i>L</i>_c for NM and M regions of the NP, respectively). The sum of b+c corresponds to the complete unfolding of the NP. The pFS-2 vector also contains an NM region, represented as a coil (a fragment of titin N2B <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Oroz1" target="_blank">[30]</a>) that can act as a spacer to avoid the noisy proximal region of the force-extension recordings, a major problem in SMFS.</p

Nanomechanical analysis of polyQ tracts and a non-amyloidogenic IDP (VAMP2).

<p>(A) Δ<i>L</i>_c (left) and <i>F</i> (right) histograms for pFS-2 polyproteins carrying polyQ tracts. The sub-threshold Q₁₉ (front row) only shows NM conformers (orange bars; represented in the force histograms below the force sensitivity of our AFM: <i>F</i>∼20 pN). Familial-disease mutations of this protein (expanded polyQs: Q₃₅ and Q₆₂) exhibit conformational polymorphism that ranges from NM conformers to M conformers (red bars), the latter class including some hM conformers (<i>F</i>≥400 pN, i.e., likely toxic conformers according to our hypothesis, see text). It should be noted that the longer the polyQ tract, the greater the conformational polymorphism and the more hM conformers found. The inhibitor QBP1 (20 µM <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Tomita1" target="_blank">[42]</a>) reduces this polymorphism and abolishes the hM conformers. The inset shows an example of a hM conformer of Q₆₂. Note that the hM class of conformers is a subset of the M set so that the percentage of hM conformers (respect to the total number of molecules sampled, <i>n</i>) is included into that of M (this also applies to the remainder main figures of this work as well as to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-t001" target="_blank">Table 1</a>). TEM images of the amyloid fibers formed by the corresponding carrier-guest proteins (not the whole pFS polyprotein) are shown on the right, highlighting the relationship between hM conformers and amyloidosis. From bottom to top, the scale bars correspond to 0.6, 0.3, 0.3, and 0.6 µm, respectively. (B) SMFS analysis of pFS-2+VAMP2. This non-amyloidogenic IDP <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Hazzard1" target="_blank">[35]</a> does not show conformational polymorphism. The scale bar from the TEM image on the right corresponds to 0.6 µm. Only data from the guest protein, and not from the carrier, are plotted in the histograms presented in this figure and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g003" target="_blank">Figures 3</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g004" target="_blank"></a><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g005" target="_blank">5</a>.</p

Nanomechanical analysis of α-synuclein.

<p>Δ<i>L</i>_c (left) and <i>F</i> (right) histograms of pFS-2 polyproteins carrying α-synuclein. The wt protein (first row) exhibits a wide-range polymorphism ranging from NM conformers (orange bars) to M conformers (red bars), including some hM conformers. Familial-disease mutations A30P and A53T increase the number of M and hM conformers of α-synuclein when compared to the wt. Treatment with QBP1 peptide (20 µM <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Tomita1" target="_blank">[42]</a>) reduces the formation of M and hM conformers in A53T α-synuclein. TEM images of the amyloid fibers formed by ubi+A53T α-synuclein are shown on the right in which amyloid fibers are clearly not formed in the presence of QBP1 (top image). From bottom to top, the scale bars correspond to 0.45 and 0.6 µm, respectively. Examples of hM conformers of A30P and A53T α-synuclein are shown in the inset.</p

Summary of the nanomechanical analysis of NPs.

<p>SMFS experiments were performed on pFS-2 polyprotein constructs, although the data reported only refer to the guest NPs. The calculation of the associated experimental errors is described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335.s018" target="_blank">Text S1</a> and they are only indicated when they are different to zero. Due to the small number of events, the frequency of hM conformers in the NPs (a subset of the M set, operationally defined with a high <i>F</i> cut off) is not statistically significant. However it correlates very well with the %M. Furthermore, statistical analysis shows that, with the exception of polyQ tracts (for which the number of M, events is too low), the differences in the %M events for NP guests are statistically significant for the following pairs: NP and non-NP, NP and familial NP, NP (except for Arc Aβ42) and NP+QBP1. These differences are not statistically significant for the following pairs: Aβ42 and Aβ42+SV111, Arc Aβ42 and Arc Aβ42+QBP1, and non NPs. The numbers in parenthesis are half of the 95% confidence interval for the %M (or NM, modeled as a Bernoulli distribution) while the numbers after ± are the SMFS experimental errors. Note that the % numbers here are the estimation for the population while the % numbers in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g002" target="_blank">Figures 2</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g003" target="_blank"></a><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g004" target="_blank"></a><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-g005" target="_blank">5</a> correspond to the raw samples.</p><p><i>n</i>, sample size. NM, no force peaks detected: <i>F</i>≤20 pN; M, at least one force peak: <i>F</i>>20 pN; hM, subset of M conformers with at least one force peak with <i>F</i>≥400 pN.</p

Nanomechanical analysis of Aβ42.

<p>Δ<i>L</i>_c (left) and <i>F</i> (right) histograms of pFS-2 polyproteins carrying Aβ42. The wt protein (first row) shows broad polymorphism ranging from NM conformers (orange bars) to M conformers (red bars). No hM conformers were found in this protein. Incubation of Aβ42 with and without SV111 (100 µM <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Arslan1" target="_blank">[38]</a>) yielded similar results, strongly indicating that our SMFS data reflect unfolding events originating from different conformations adopted by the monomeric forms of NPs (as opposed to intermolecular interactions of the oligomeric species). Arc Aβ42 (E22G) familial-disease mutation increases the number of M and hM conformers when compared to the wt protein. Incubation with QBP1 peptide (20 µM <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio.1001335-Tomita1" target="_blank">[42]</a>) does not impair the formation of M conformers in Arc Aβ42. For F19S/L34P Aβ42 only NM conformations were observed despite the larger sample size (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001335#pbio-1001335-t001" target="_blank">Table 1</a>). TEM images of the amyloid fibers formed by the corresponding proteins are shown on the right. From bottom to top, the scale bars correspond to 0.6, 0.6, 0.45, 0.35, and 0.9 µm, respectively. An example of hM conformer of Arc Aβ42 is shown in the inset.</p