Influence of TTR in A-Beta aggregates.

<p>A-Beta alone was incubated for 3 hours at 37°C to form aggregates and then further incubated with or without TTR for another 3 hours. Analysis was done by western blot following separation under denaturing conditions. Results indicated that in preparations of A-Beta incubated with TTR, both bands corresponding to A-Beta monomer and to the higher molecular form presented decreased intensities, as compared to the same bands in the A-Beta alone preparation.</p

A-Beta proteolysis by TTR analyzed by mass spectrometry.

<p>A- Preparations of A-Beta alone, A-Beta incubated with TTR (A-Beta+TTR), or TTR alone incubated for 3 hours at 37°C, analyzed by MALDI-TOF/TOF mass spectrometry. The new 1698 Da peak present in A-Beta+TTR preparations was submitted to MS/MS peptide de novo sequencing, and showed to correspond to the first 14 aminoacid residues of A-Beta peptide. B- Fraction corresponding to peak c of A-Beta incubated with TTR for 15 hours at 37°C, and subjected to RP-HPLC, and also analyzed by mass spectrometry, showed a peak of approximately 3094.7 Da which indicates the presence of A-Beta peptide 1–27.</p

Kinetics of A-Beta proteolysis by TTR.

<p>2 µg of A-Beta were incubated alone (−) or in the presence of 15 µg of human TTR (+), either isolated from sera (right panels) or recombinant (left panels), for different periods of time: 1 hour (1 hr), 3 hours (3 hrs), 6 hours (6 hrs) and 15 hours(15 hrs), at 37°C. Visualization was performed either by staining the SDS-PAGE gel with Coomassie Blue (upper panels) or by western blot of the gel using an antibody specific for A-Beta (lower panels). Bands corresponding to the TTR and A-Beta (1–42) monomers are indicated by arrows (left side) as well as the molecular weight standards used (right side); TTR incubated for 15 hrs at 37°C was also run on the gel (TTR); MW- molecular weight standards.</p

A-Beta proteolysis by TTR is KPI-sensitive.

<p>A- A-Beta incubated with TTR (A-Beta+TTR) shows a weaker A-Beta monomer band as compared to A-Beta alone (A-Beta), indicative of proteolysis, as analyzed by SDS-PAGE electrophoresis followed by western blot. Pre-incubation of TTR with pefabloc (A-Beta+(TTR+pefabloc)) and with an αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI⁺−APP)) inhibits TTR proteolytic activity, whereas the αAPP peptide without the KPI domain (A-Beta+(TTR+KPI⁻−APP)) facilitates proteolysis. B- % of inhibition of TTR proteolysis by quantification of band intensity in A. C- Ultrastructural analysis by TEM of preparations incubated for 15 hours, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002899#s2" target="_blank">Materials and Methods</a>. TTR inhibited A-Beta aggregation as compared with A-Beta incubated alone (upper panels). Pre-incubation of TTR with αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI⁺−APP)) abrogated TTR ability to avoid A-Beta aggregation, whereas αAPP lacking the KPI domain (A-Beta+(TTR+KPI⁻−APP)) did not affected TTR activity (lower panels). Scale bar = 500 nm.</p

A-Beta peptide cleavage by TTR.

<p>A- RP-HPLC profiles of A-Beta proteolysis by TTR. 40 µg A-Beta was incubated with TTR (192 µg) for different periods of time (3, 6, and 15 hours). Enzymatic hydrolysis was stopped and samples were subjected to RP-HPLC analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002899#s2" target="_blank">Material and Methods</a> section. B- A-Beta (1–42) aminoacid sequence and the cleavage sites of A-Beta by various enzymes including NEP (N), IDE (I), ACE (A), ECE (E), plasmin (P) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002899#pone.0002899-Wang2" target="_blank">[25]</a> and TTR (T).</p

Amyloidogenic potential of A-Beta peptides (1–14) and (15–42).

<p>A- Th T binding assay of A-Beta peptides; A-Beta (1–14) does not bind Th T whereas peptide (15–42) presents amyloidogenic potential which is lower than the (1–42) counterpart. Results were presented by plotting the intensity of fluorescence at 450 nm which is the characteristic novel excitation maxima formed upon Th T binding for each peptide; AU-arbitrary units. * p<0.02; ** p<0.0006. B- Ultrastructural analysis by TEM of A-Beta peptides incubate for 5 days at 37°C. Peptide (1–14) did not form amyloid fibrils; peptide (15–42) formed fibrils under these circumstances but shorter and less abundant than A-Beta (1–42). Scale bar = 500 nm.</p

TTR spheroid oligomers and protofibrils.

<p><b>A.</b> 1×1 µm² AFM height contrast image of a mixed population of spheroid oligomers and short protofibrils. Black arrows point out examples of spheroid oligomers with various shapes and sizes. <b>Inset,</b> magnified view of a protofibril displaying a stack-like arrangement of flat, disc-shaped oligomers reminiscent of annular origin. <b>B.</b> 1×1 µm² AFM height contrast image of a mixed population of spheroid oligomers and longer protofibrils. Black arrows point out examples of spheroid oligomers with various shapes and sizes. <b>Inset</b>, magnified view of a protofibril in which the underlying periodic structure is probably helical. <b>C.</b> Topographical molecular volume histogram of 341 (<i>n</i>) spheroid TTR oligomers. The numbers above the modes correspond to the mean values of gaussian fits.</p

Formation and disappearance of annular oligomers.

<p><b>A.</b> Dynamic light scattering spectra of native TTR and at given time points after the start of acidification where the emergence of a small population of larger particles follows a trend towards smaller apparent hydrodynamic radii (Rh_app). <b>B.</b> Time course of the apparent size of the different populations during aggregation and their corresponding weighted average. The arrows indicate the times points where images shown in C and D were taken. <b>C & D.</b> AFM images (phase contrast) of particles taken at 9 and 12 h respectively and where annular oligomers (C) and spheroid (D) oligomers can be observed. The inset represents a 50×50 nm topography image of the corresponding samples (height scale up to 2.5 nm) <b>E.</b> Height-contrast AFM image of annular oligomers undergoing transitions. <b>E.</b> Magnified view of fusing annular oligomers indicated in <i>D</i>. Height, amplitude and phase contrast images (left to right) are shown. Scale bar, 10 nm.</p

Disassembly of TTR protofibrils.

<p><b>A.</b> AFM height contrast image recorded after 1 minute of sample dilution into PBS. <b>B.</b> AFM height (top) and amplitude (bottom) contrast images recorded after 5 minutes of sample dilution into PBS. <b>C.</b> AFM height (top) and amplitude (bottom) contrast images recorded after 15 minutes of sample dilution into PBS. <b>Insets</b>, magnified image of a single annular oligomer (left) and a laterally-associated doublet of annular oligomers (right). <b>D.</b> Distribution of the diameter of annular oligomers observed during assembly (yellow) and disassembly (purple). <b>E.</b> Distribution of the topographical height of annular oligomers observed during assembly (yellow) and disassembly (purple).</p

Model of TTR protofibril assembly and disassembly.

<p>Relevant dimensions and periodicity parameters of the intermediates are indicated where applicable. Length of the arrows scale with the hypothesized transition kinetics.</p