RecPrP^res aggregates are similarly neurotoxic as PrP27-30.

<p>RecPrP^res aggregates were produced by incubation for 24 hrs with 400 mM NaF followed by PK-digestion for 1 hrs at 37°C. 100, 50 and 25 nM of dialyzed recPrP^res aggregates were added to the medium of 1×10⁵ N2A neuroblastoma cells and cell viability was measured after 24 hrs of incubation using the MTT assay. As a negative control, the same volume of PBS was added to the well (control). Purified PrP27-30 from RML infected mice brain, soluble recPrP (recPrP) and the reaction buffer without protein (buffer) were also assayed as controls. All experiments were done in triplicate and the values correspond to the average ± standard error. The reduction of cell viability produced by addition of recPrP^res or PrP^Sc was highly significantly (P<0.001) different from soluble recPrP and the buffer control, as determined by student t-test.</p

Seeding-nucleation model of protein aggregation and the cross-seeding phenomenon.

<p>(A) Amyloid aggregates are formed following the seeding-nucleation polymerization model. This aggregation process is divided into two phases, the so-called nucleation/lag phase and the polymerization/elongation phase (solid lines). Since nuclei are formed, the aggregation increases in an exponential manner from small oligomers to fibers. The addition of preformed seeds leads to a shorter lag phase and a faster aggregation (dashed lines). (B) Seeding can occur by adding a previously formed seed, facilitating and speeding up the polymerization process. These seeds can have the same chemical nature as the nuclei, leading to a homologous seeding, or be made from a different protein, inducing a heterologous seeding or cross-seeding.</p

Kosmotropic anions induce formation of PrP^Sc-like protease-resistant species.

<p><b><i>A–F:</i></b> RecPrP was incubated with different concentrations of sodium sulfate (Na₂SO₄, <b><i>A</i></b><i>)</i>, sodium chloride (NaCl, <b><i>B</i></b>), tetramethylammonium sulfate (((CH₃)₄N)₂(SO₄), <b><i>C,E</i></b>) and tetramethylammonium chloride ((CH₃)₄N(Cl), <b><i>D,F</i></b>) as described in Experimental Procedures, followed by Western Blotting (<b><i>A–D</i></b>) or silver staining (<b><i>E</i></b><b>,</b><b><i>F</i></b>). Salts concentrations (mM) for the reactions were 0, 100, 200, 300, 400 and 500 for lanes 1, 2, 3, 4, 5 and 6, respectively. Undigested recPrP standard is shown on lane 7 for each figure. The arrows indicate the signal corresponding to proteainse K (PK).</p

Structural properties of recPrP aggregates.

<p><b><i>A:</i></b> Buffer and baseline-corrected FTIR spectra of PK-treated salt-induced recPrP aggregates (solid line) compared to those of soluble recPrP (dashed line) and PrP27-30 purified from the brain of mouse infected with RML prions (dotted line). FTIR spectra were obtained using the conditions described in Experimental Procedures. <b><i>B:</i></b> To study the morphology of the PK-treated recPrP aggregates, samples were loaded onto EM grids, stained with silver nitrate and visualized under TEM. Representative images for both PrP27-30 and recPrP^res aggregates are shown at two different magnifications (see the magnification bars).</p

Potential mechanisms for recPrP aggregation induced by kosmotropic salts.

<p>The soluble monomeric recPrP is represented as a two-domain protein, C-terminal globular domain (triangle) containing a large proportion of alpha-helical structure and the natively-unfolded N-terminal domain (solid line). Misfolded recPrP acquires intermolecular beta-sheet secondary structure in the natively unfolded region 90–145 (represented as a dark gray horizontal rectangle). The structural fate of the C-terminal domain is unclear, but most likely involves a conformational rearrangement (represented as a dark blue rectangle). The kosmotropic anions are represented by the blue circles. The dotted arrows indicate a misfolding process, while the solid arrows represent the protease digestion reaction (PK). Three putative models to explain the effect of the salt on inducing the formation of recPrP^res are proposed. <b><i>A:</i></b> Binding of kosmotropic anions may occlude protease cleavage sites within the N-terminal domain of PrP. However, this model per se does not account for the partial resistance to proteolytic degradation of the whole C-terminal domain as well as for the structural changes induced by salt. <b><i>B</i></b><b>:</b> A salting-out-like mechanism locally increases PrP concentration in a native-like conformation, followed by either an induction or acceleration of misfolding to form intermolecular beta-sheets giving rise to PrP^Sc-like aggregates. <b><i>C</i></b><b>:</b> A combined effect of high concentrations of kosmotropic anions that partially salt-out recPrP in a close-to-native fold from the bulk solution, along with specific anion binding to PrP that further stabilizes the N-terminal domain induce a protease-resistant recPrP conformation with PrP^Sc features.</p

Time-dependent formation of recPrP^res.

<p><b><i>A:</i></b> RecPrP was aggregated in 400 mM NH₄F for different times: 0 hrs (lane 1), 3 hrs (lane 2), 10 hrs (lane 3), 24 hrs (lane 4), 72 hrs (lane 5) and 144 hrs (lane 6) and the recPrP^res product was analyzed by silver staining. PK signal is highlighted by the solid arrow. The undigested recPrP signal is indicated by the dashed arrow. In all panels, samples were digested using PK at 1/10 PK/recPrP ratio for 1 hrs at 37°C and then subjected to silver staining. Molecular weights markers (kDa) are shown on the left side. <b><i>B:</i></b> A similar reaction was followed by the increase in Th-T signal in time. Each time point corresponds to the mean and standard error of 3 independent replicates. The points fit very well to a sigmoidal curve (dashed line).</p

Formation of protease-resistance recPrP aggregates (recPrP^res).

<p><b><i>A-B:</i></b> RecPrP was incubated with different concentrations of NH₄F (<b><i>A</i></b>) and NaF (<b><i>B</i></b>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031678#s4" target="_blank">materials and methods</a>, followed by Western Blotting. Salts concentrations (mM) for the reactions were 0, 100, 200, 300, 400 and 500 for lanes 1, 2, 3, 4, 5 and 6, respectively. A small amount of undigested recPrP used as a marker of electrophoretical migration is shown on lane 7 for each figure. <b><i>C</i></b>: Antibody mapping analysis of protease resistance fragments was performed using a 400 mM NH₄F-based reaction incubated for 24 hrs. Duplicated samples at two different dilutions (1/2 and 1/1 per left and right lane, respectively) were western-blotted using monoclonal antibodies 6D11 and M-20. The arrow indicates the presence of oligomeric species. <b><i>D:</i></b> RecPrP was incubated for 0 hrs (dashed line) or 24 hrs (solid line) with 400 mM NH₄F and then fluorescence emission spectra of samples in the presence of 10 uM Th-T was recorded. An emission maximum was obtained at 491 nm when excited at 435 nm, typical of amyloid-like aggregates.</p

Automation and screening.

<p>The complete assay was automated and performed in a similar way mimicking all steps of the actual screening. 100% DMSO was used to replace the compound. (a) Two full 384 plates were used. Plate 1 and 2 yielded a Z score of 0.91 and 0.81; % CV 13.99 and 15.12 for the full reaction, respectively. (b) Z scores of 4 pilot plates screened in duplicate using this automated assay format.</p

Chemical structure and formula of identified hits.

<p>Chemical structures, masses and formulas of the inhibitors are listed in the tabular format.</p

Characterization of enzyme activity.

<p>(a) In order to test the effect of pH on CaN activity, CaN assay was performed at different pH. Our data suggested that pH 7 is optimum for CaN activity. To study the effect of bivalent metal ions on CaN activity, enzyme activity assays were performed at different MnCl₂ (b) and MgCl₂ (c) concentrations. The data indicated that bivalent manganese increased CaN activity almost two times (b) whereas magnesium had not effect (c). Note that (a), (b) and (c) were performed in absence of CM. (d) The concentration of CM which produces 50% activation (IC₅₀ CM) of CaN was determined by plotting initial reaction velocities at different Ca²⁺ and CM concentrations. Our data indicated that CM produces half maximum CaN activity at 1:1 molecular ratio. (e) Enzyme K_m was determined by plotting initial velocity at different substrate (RIIP) concentrations. The K_m was 213 μM. (f) DMOS tolerance of the enzyme was assayed by performing the assay at different DMSO concentration ranging from, 0.125 to 4%. Our data indicate that at ≤0.5% DMSO did not alter enzyme activity. The assays were done in duplicate (except for experiment in Fig 1d) and the data were expressed as means and standard error. The fittings were done using Prism software.</p