A Systematic Investigation into the Effect of Protein Destabilisation on Beta 2-Microglobulin Amyloid Formation

Beta-2-microglobulin (ß2m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (1 µm), straight morphology are produced by incubation of the protein at pH=3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of ß2m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states

Fungal hydrophobins.

<p>Fungal hydrophobins are unique amphipathic proteins with multiple roles in the fungal life cycle and in mediating interactions between fungus and host. There is diversity in the primary sequences of hydrophobins but they share a similar core three-dimensional structure and a pattern of four disulfide bonds (shown in amber) that stabilize the structures. Increasingly, these proteins show potential for modification of hydrophobic nanomaterials and in solubilizing lipophilic drugs.</p

Common core structure of amyloid fibrils by synchrotron X-ray diffraction

Tissue deposition of normally soluble proteins as insoluble amyloid fibrils is associated with serious diseases including the systemic amyloidoses, maturity onset diabetes, Alzheimer's disease and transmissible spongiform encephalopathy. Although the precursor proteins in different diseases do not share sequence homology or related native structure, the morphology and properties of all amyloid fibrils are remarkably similar. Using intense synchrotron sources we observed that six different ex vitro amyloid fibrils and two synthetic fibril preparations all gave similar high-resolution X-ray fibre diffraction patterns, consistent with a helical array of ß-sheets parallel to the fibre long axis, with the strands perpendicular to this axis. This confirms that amyloid fibrils comprise a structural superfamily and share a common protofilament substructure, irrespective of the nature of their precursor proteins

Solid-State NMR Structure Determination from Diagonal-Compensated, Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints

We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and ¹H back-exchanged proteins. To achieve this, we use homonuclear ¹H–¹H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal

Additional file 1 of iPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype

Additional file 1: Supplementary Methods. Table S1. Primary antibodies used for immunofluorescence. Table S2. Secondary antibodies used for immunofluorescence. Fig. S1. Sanger sequencing chromatograms showing APOE genotyping of codon 112 (rs429358) and codon 158 (rs7412) for all iPSC lines. Yellow highlight indicates the position of the single nucleotide polymorphism. Fig. S2. Immunofluorescence images of iPSCs from three healthy control lines (Ctrl-06, Ctrl-71, Ctrl-88) and three familial AD lines harbouring a PSEN2 (N141I) mutation (fAD-08, fAD-948, fAD-950). The cells were stained for pluripotency markers Nanog (red), Oct 3 (green) and all nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. Fig. S3. Immunofluorescence images of iPSC-derived NPCs from three healthy control lines (Ctrl-06, Ctrl-71, Ctrl-88) and three familial AD lines harbouring a PSEN2 (N141I) mutation (fAD-08, fAD-948, fAD-950). The cells were stained for A the neural progenitor markers Pax-6 (red) and Nestin (green), B a pluripotency marker Oct3 (green) and all nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. Fig. S4. Immunofluorescence images of iPSC-derived astrocytes from three healthy control lines (Ctrl-06, Ctrl-71, Ctrl-88) and three familial AD lines harbouring a PSEN2 (N141I) mutation (fAD-08, fAD-948, fAD-950). The cells were stained for A astrocyte markers GFAP (red) and S100β (green), B the NPC marker nestin (green). All nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. Fig. S5. Transcriptomic analysis of iPSC-derived cell types and primary human fetal astrocytes. A Principal component analysis and B cluster analysis of iPSC-derived astrocytes (black) from healthy control lines (lines 06, 71 & 88) generated in our study and commercially-available primary astrocytes grown in our lab (green) combined with a datasets from Tcw et al (23), including primary astrocytes (purple), iPSC-derived NPCs (light blue), astrocytes (dark blue) and neurons (yellow). Contrast matrix of differential gene expression between cell types comparing datasets C our iPSC-derived astrocytes vs. Tcw et al’s primary astrocyte and iPSC-derived neurons datasets, D Tcw et al’s dataset alone, E our data alone. Fig. S6 Immunofluorescence images of iPSC-derived microglia-like cells from three healthy control lines (Ctrl-06, Ctrl-71, Ctrl-88) and three familial AD lines harbouring a PSEN2 (N141I) mutation (fAD-08, fAD-948, fAD-950). Images show cells stained for A the microglial markers IBA1 (red), TREM2 (green), B CX3CR1 (red) and all nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. Fig. S7. Multi-cytokine array of Alzheimer’s or healthy iPSC-derived astrocytes A basally and B after 24 h exposure to 10 μM Aβ42 and iPSC-derived microglia-like cells C basally and D after 24 h exposure to 10 μM Aβ42. The figure displays the mean ± SD of three cell lines with the average of two experimental duplicates per line. Multiple unpaired, non-parametric Mann-Whitney t-tests adjusting for a 0.05 false discovery rate were used to test whether there were statistically significant differences between mean cytokine/chemokine release of AD-derived and healthy control astrocytes and microglia-like cells (*p < 0.05, **p < 0.01). Cytokines that yielded an average intensity value less than 10% of the maximum (represented by the dotted line) were considered background and not included in the statistical analysis. Fig. S8. Multi-cytokine array of APOE ε3/ε3 and APOE ε3/ε4 iPSC-derived astrocytes A basally and B after 24 h exposure to 10 μM Aβ42 and iPSC-derived microglia-like cells C basally and D after 24 h exposure to 10 μM Aβ42. The figure displays the mean ± SD of three cell lines with the average of two experimental duplicates per line. Multiple unpaired, non-parametric Mann–Whitney t-tests adjusting for a 0.05 false discovery rate were used to test whether there were statistically significant differences between mean cytokine/chemokine release of APOE ε3/ε3 and APOE ε3/ε4 astrocytes and microglia-like cells (*p < 0.05). Cytokines that yielded an average intensity value less than 10% of the maximum (represented by the dotted line) were considered background and not included in the statistical analysis. Fig. S9. Concentrations of A Aβ42, B Aβ40, C the ratio of Aβ42:40 and D total Aβ protein quantified from iPSC-derived astrocyte supernatants 72 h after plating. Secreted Aβ concentrations were measured using a highly sensitive ELISA and normalised to total protein concentration determined by BCA. The figure displays the mean ± SD of three cell lines with n ≥ 2 independent experiments per line. A post hoc unpaired t-test was used to test whether there were statistically significant differences between mean of APOE ε3/ε3 and APOE ε3/ε4 astrocytes

A Coumarin-Based Array for the Discrimination of Amyloids

Self-assembly of misfolded proteins can lead to the formation of amyloids, which are implicated in the onset of many pathologies including Alzheimer’s disease and Parkinson’s disease. The facile detection and discrimination of different amyloids are crucial for early diagnosis of amyloid-related pathologies. Here, we report the development of a fluorescent coumarin-based two-sensor array that is able to correctly discriminate between four different amyloids implicated in amyloid-related pathologies with 100% classification. The array was also applied to mouse models of Alzheimer’s disease and was able to discriminate between samples from mice corresponding to early (6 months) and advanced (12 months) stages of Alzheimer’s disease. Finally, the flexibility of the array was assessed by expanding the analytes to include functional amyloids. The same two-sensor array was able to correctly discriminate between eight different disease-associated and functional amyloids with 100% classification

Surface-Induced Hydrophobin Assemblies with Versatile Properties and Distinct Underlying Structures

Hydrophobins are remarkable proteins due to their ability to self-assemble into amphipathic coatings that reverse surface wettability. Here, the versatility of the Class I hydrophobins EASΔ15 and DewY in diverse nanosuspension and coating applications is demonstrated. The hydrophobins are shown to coat or emulsify a range of substrates including oil, hydrophobic drugs, and nanodiamonds and alter their solution and surface behavior. Surprisingly, while the coatings confer new properties, only a subset is found to be resistant to hot detergent treatment, a feature previously thought to be characteristic of the functional amyloid form of Class I hydrophobins. These results demonstrate that substrate surface properties can influence the molecular structures and physiochemical properties of hydrophobin and possibly other functional amyloids. Functional amyloid assembly with different substrates and conditions may be analogous to the propagation of different polymorphs of disease-associated amyloid fibrils with distinct structures, stability, and clinical phenotypes. Given that amyloid formation is not required for Class I hydrophobins to serve diverse applications, our findings open up new opportunities for their use in applications requiring a range of chemical and physical properties. In hydrophobin nanotechnological applications where high stability of assemblies is required, simultaneous structural and functional characterization should be carried out. Finally, while results in this study pertain to synthetic substrates, they raise the possibility that at least some members of the pseudo-Class I and Class III hydrophobins, reported to form assemblies with noncanonical properties, may be Class I hydrophobins adopting alternative structures in response to environmental cues

Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates.

Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer's and Parkinson's disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases