Misfolding of HLS prevents oligomerization.

<p>(A) Far-UV CD data for the thermally-induced denaturation of the native and refolded protein SBD556 (red and purple circles, respectively) and SBD641 (green and brown, respectively); a three-state denaturation model was used to analyse the experimental data (continuous lines; see Materials and Methods and Table S3 in File S1). (B) Tryptophan (W580) fluorescence spectra of SBD641 in their native (green line), thermally denatured (black dashed line) and refolded (brown continuous line) states; the spectrum of SBD556 in its native state is shown as a negative control (red continuous line). The calculated spectrum of SBD641, corresponding to 80% of the protein molecules in the native structure and 20% of the molecules in the thermally denatured state, assuming that the total fluorescence signal at a given wavelength is a linear combination of the fluorescence signal for each populated state (brown dashed lines), is also shown for comparison with the spectrum of the protein in its refolded state. (C) Analytical SEC performed on native and refolded (after thermal denaturation of the protein) states of SBD641 (green and brown lines, respectively). The molecular weights of the standard proteins used to calibrate the column are reported at the top of the chromatogram.</p

The interdomain linker is essential for chaperone oligomerization.

<p>(A) Far-UV CD spectrum of native ∆LSBD641 (black) compared to that of the SBD641 variant (green). (B) Comparison of the SEC chromatograms obtained for SBD641 (in green) and ΔLSBD641 (in black) at a protein concentration of 6 µM. (C) nESI MS analysis of ΔLSBD641 (monomer in green, dimer in red). (D) Comparison of the SEC chromatograms obtained for FL-Hsp70 (in black) and FL-Hsp70 2LD (in cyan) at a protein concentration of 220 µM. The molecular masses of the standard proteins are again reported at the top of the chromatogram.</p

The different protein variants have different propensities to oligomerize.

<p>Analytical SEC and nESI mass spectra of the different protein constructs measured at the same protein concentration (6 µM): FL-Hsp70 (A, C), SBD641 (B, D), SBD556 (E, G) and C-term (F, H) variants. The SEC elution profiles were fitted to multi-peak Gaussian functions to evaluate the relative fractions of monomeric and oligomeric protein species. The molecular masses of the standard proteins used to calibrate the SEC column are reported at the top of the chromatogram. The elution peaks in SEC and the peaks in MS (Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067961#pone.0067961.s001" target="_blank">File S1</a>) corresponding to monomeric protein are coloured in green for all the variants, while those for dimers are in red, trimers in blue and tetramers in yellow. Note that comparing visually the SEC and MS data quantitatively is difficult as the relative peak heights in the mass spectra report on molecular abundance, which is independent of oligomeric state, while the SEC data depend on molecular absorbance.</p

Substrate binding affects the oligomerization equilibrium.

<p>(A) Fluorescence titration of the binding of the NR peptide to the different Hsp70 constructs (showing the fraction of bound substrate as a function of chaperone concentration): SBD556 (red circles), native (green circles) and refolded (brown circles) SBD641, ΔLSBD641 (black circles), and FL-Hsp70 (blue circles). The continuous lines represent the best fits of the data to a single binding site model. (B) SEC analysis of SBD641 in the absence (green line) and the presence (orange line) of a 14-fold excess of NR peptide. (C) nESI MS spectrum of SBD641 in the presence of the NR peptide at a protein:peptide ratio of 1:1 at a sample cone voltage of 60 V. Inset is the region of the spectrum showing dimers, at increasing sample cone voltages from front to back. The coloured circles represent charge states that correspond to the same protein species: uncomplexed monomeric protein in dark green, monomeric SBD641 complexed with the NR peptide in light green, the uncomplexed dimeric protein in red, dimeric SBD641 complexed with a single NR peptide molecule in pink, and dimeric SBD641 complexed with two NR peptide molecules in orange. The protein: peptide molar ratio used allowed sufficient resolution of the various charge state series for unambiguous assignment of substrate-bound chaperone states, which are validated by the successive removal of peptide equivalents upon increasing collisional activation (inset). The spectra shown are representative of five repeats, each incorporating >150 scans, with the relative abundances of apo- and holo-forms varying by <5% between individual nanoESI injections. Observed masses differ from those expected, due to residual adduction of solvent molecules and buffer ions, by <1%, as is typical in the spectra of non-covalent complexes (Table S6 in File S1) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067961#B57" target="_blank">57</a>].</p

Model for Hsp70 oligomerization.

<p>Hsp70 is composed of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD), which are connected to each other by a highly conserved hydrophobic linker (see insert). The SBD is in turn divided into two subdomains: the substrate-binding subdomain (SBSD), with mostly β-sheet structure, and the helical lid subdomain (HLS) that is rich in α-helical structure. The schematic images of the Hsp70 molecule are based on the crystal structure of DnaK complexed with ADP and substrate (pdb: 2KHO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067961#B48" target="_blank">48</a>]). In the absence of nucleotide and of a polypeptide substrate, Hsp70 exists at equilibrium as a distribution of monomers (state A), dimers (state B), and higher order oligomers (state C). In the monomeric state, the two functional domains are allosterically independent and the HLS of the SBD is highly mobile. Our results show that chaperone oligomerization occurs through an interaction between the HLS of one protein molecule and the interdomain linker of another molecule (this interaction is shown by an arrow in state B), allowing the formation of oligomeric species. In the presence of an unfolded polypeptide substrate, however, the equilibrium between the different states of the chaperone is shifted towards the accumulation of monomeric protein, which is the state that shows the highest affinity for substrates because of the stabilization of the complex by the HLS that acts as a lid preventing substrate release (state D). The oligomers, however, have only one HLS in each species that is free from interactions with the linker of another protein molecule, and are therefore able to stabilize the binding of only one substrate molecule; this protein molecule will, however, be able to stabilize the bound substrate (state E). The remaining HLSs interact with another molecule of the chaperone, and therefore they are unable to act as a lid to their corresponding substrate-binding pockets, resulting in a decreased affinity for substrate (state F). As a consequence of this fact, the overall affinity for the substrate per chaperone molecule is decreased in the oligomeric species when compared with the monomeric protein. Indeed, the higher the order of the oligomer, the higher the number of low-affinity binding pockets per chaperone molecule and the lower the overall affinity of the chaperone molecule for the substrate. Once the substrate folds (to give the native state), its hydrophobic residues are no longer able to interact with the binding pocket of the chaperone, which results in substrate release and the chaperone recovers its initial state (an equilibrium between states A, B and C).</p

Local Cooperativity in an Amyloidogenic State of Human Lysozyme Observed at Atomic Resolution

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein

ELN484228 provides protection in cellular models of αSyn-mediated dysfunction.

<p><b>A.</b> ELN484228 alleviates αSyn-mediated impairment of vesicular dynamics. H4 neuroglioma cells over-expressing αSyn from a tetracycline inducible promoter were cultured for 24 hours in the absence or presence of 1 µg/ml tetracycline to induce αSyn and ELN484228 or control compound ELN484217 (compound number 38 in table S4 in Supporting Information text <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087133#pone.0087133.s001" target="_blank">file S1</a>). Open bars: without compound, black bars: with indicated amount of compound. Cells were assayed for phagocytic activity as a measure of αSyn-mediated impairment of vesicular function. 4 μ beads were added for 90 minutes and a phagocytic index was calculated by microscopic visualization. Each sample was run in triplicate and experiments were run three independent times. The phagocytic indices for each individual experiment were averaged and statistical analyses run on the final averages from the three experiments. T-test analysis of the combined averages of the three experiments revealed a significant difference in phagocytosis between Tet-induced samples with and without ELN484228 (n = 3+/− s.e.m *p≤0.001 versus no compound Tet-induced sample). <b>B.</b> Microglia isolated from postnatal day 1 to 3 pups from hSNCA^E46K transgenic (αSyn ) or non-transgenic littermates were incubated for 24 hours with 100 µM ELN484217 or ELN484228 followed by addition of 10 µm beads for 90 minutes. A phagocytic index was calculated by microscopic visualization (n = 3+/− s.e.m *p≤0.001). <b>C.</b> ELN484228 alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced (‘control’) or transduced with adenovirus encoding A53T αSyn, in the absence or presence of 10 µM ELN484228. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. The lengths of neurites staining positive for both MAP2 and TH were measured using the NIS-Elements software. Data are plotted as the mean ± s.e.m. n = 3 for neuron viability analysis; n = 160–206 for neurite length analysis. *p-value≤0.05, ***p-value≤0.001 versus aSyn A53T virus in the absence of compound; one-way ANOVA with Newman-Keuls post-test. <b>D.</b> ELN484228 reduces translocation of αSyn to the phagocytic cup<b>.</b> To assess αSyn translocation, H4 cells were treated with 100 µM ELN484228 and 1 µg/ml tetracycline for 24 hours; cells were then stimulated with 4 μ beads for 90 minutes. Samples were fixed and stained with 5C12 antibody to detect αSyn (red). Cells were counterstained with 488-phalloidin (green) and Hoechts (blue). A dotted circle indicates the position of the bead. <b>E.</b> ELN484228 reduces translocation of αSyn to synapses. Rat hippocampal neurons (∼21DIV) grown in serum-free conditions were treated for 24 hours with 1 µM ELN484228 or 0.01% DMSO vehicle. On the left side is a representative confocal microscopic image showing localization of αSyn (red) detected with 5C12 antibody, and localization of the presynaptic marker synaptophysin (green). Scale bar is 5 µm. Images were subjected to quantitative analysis and synaptic αSyn levels were determined as the amount of signal that colocalizes with the synaptic synaptophysin marker. Automated measurements were performed in Metamorph imaging analysis software to determine synaptic αSyn and synaptophysin levels by integrated intensity or pixel area, respectively. Values represent mean +/− SEM, n = 1000 terminals (αSyn) or 18 optical fields (synaptophysin) per condition, and derived from 2–3 independent cultures. Quantitation demonstrates that ELN484228 reduces the synaptic levels of αSyn in rat hippocampal neurons.</p