Electron cryomicroscopy analysis of infectious prion protein amyloid fibrils.

<p><b>(A)</b> Section of a cryo electron micrograph showing prion fibrils lacking the glycosylphosphatidylinositol (GPI) anchor. A single isolated and twisted fibril used for the 3-D reconstruction is enclosed by a black box. <b>(B)</b> Close-up view of the isolated prion fibril. <b>(C)</b> Reprojected image of the 3-D fibril map for comparison with the unprocessed image (B). <b>(D)</b> 3-D reconstruction of the GPI-anchorless prion fibril. <b>(E)</b> Cross section of the reconstructed fibril showing two distinct protofilaments. <b>(F)</b> Contoured density maps of the cross section with lines contoured at increasing levels of 0.125 σ. <b>(G)</b> Cartoon depicting the proposed configuration of the polypeptide chains in the prion fibril. Please note that this is not an atomistic model. <b>(H)</b> Close-up view of the possible ß-sheet stacking in a four-rung ß-solenoid architecture for illustration purposes only. Different colors represent different ß-solenoid rungs. Characteristic distances of the four-rung ß-solenoid architecture are labeled. Figure adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006229#ppat.1006229.ref010" target="_blank">10</a>].</p

Infectious prions and prion-like proteins.

<p>Infectious prions and prion-like proteins.</p

Identification of a TLQP-21 binding protein from SH-SY5Y cell homogenates.

<p>A ~70 kDa protein was bound on the biotin-TLQP-21 treated/attached column (fractions TF 4–5) but not in the corresponding fractions eluted from the control column (<i>i</i>.<i>e</i>., from a sample prepared in the same way but subjected to chromatography on an affinity column which had not been pre-loaded with biotin-TLQP-21:CF4-5). The eluted fractions were resolved by SDS-PAGE and analysed by SYPRO^® Ruby Protein Gel Stain.</p

Characteristics of the interactions involved in TLQP-21 recognition by HSPA8.

<p>A: Details of the hydrogen bonding interactions between TLQP-21 and HSPA8. HSPA8 and TLQP-21 are colored as salmon and lime-green color, respectively. B: Variations of the number of hydrogen bonding between TLQP-21 and HSPA8 with simulation time. C: Electrostatic surface potential of HSPA8 when TLQP-21 is bound to the protein. Regions of positively and negatively charged surface area are colored in blue and red color, respectively. Arginine residues of TLQP-21 are shown in blue stick mode.</p

TLQP-21 binds to HSPA8 expressed on the cell surface of live SH-SY5Y cells.

<p>Membrane proteins, following biotinylation, were applied onto the monomeric avidin column. The eluted proteins (T) were analysed by the Western blot using anti-HSPA8 antibody; the control sample (C) was obtained from cells treated under the same conditions, but no TLQP-21 was added.</p

Structures of the HSPA8-TLQP-21 obtained from molecular docking study.

<p>HSPA8 is colored in green whereas the bound TLQP-21 is colored in pink.</p

MASCOT analysis of proteins present in the ~70kDa band detected after affinity chromatography of SH-SY5Y cell homogenates using TLQP-21 as a “bait”.

<p>MASCOT analysis of proteins present in the ~70kDa band detected after affinity chromatography of SH-SY5Y cell homogenates using TLQP-21 as a “bait”.</p

Dynamic Mass Redistribution analysis of TLQP-21 interaction with HSPA8.

<p>A range of different concentrations of peptide were tested (6.25, 12.5, 25, 50, 100, 150 and 200 μM) in triplicates. Responses are shown at 20 (A) and 60 (B) minutes obtaining an K_d of 91.56 and 93.26 μM, respectively. Label-free responses are measured as shifts in reflected wavelength and are expressed in picometers (pm).</p

Kinetics of PK digestion of unpurified GPI

<p>⁻<b>PrP^Sc.</b> Samples were digested with PK (25 µg/ml) and the reaction stopped after 0, 30, 60, 120, 180, 240, 300 and 360 minutes. Samples were treated with PNGase F and subjected to Tricine-SDS-PAGE the blot was probed with R1 antibody.</p

MALDI-TOF spectrum of PK-treated purified GPI

<p>⁻<b>PrP^Sc.</b> Doubly-charged ions from peptides with m/z 16371 and 17148 are indicated (*). Low resolution in the >16 kDa region precluded identifying unmarked peaks. A scheme of GPI^- PrP sequence with PK cleavage points (color coded) and secondary structure of PrP^C is included at the top: (octarepeats (□), β-sheets (▸), and α-helices (∥)); epitopes of the mAbs used are also indicated.</p