Publications

Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type-amino acid (AA) substitutions-despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate a comprehensive atlas of diverse variant effects for a disease protein, the amyloid beta (Aβ) peptide that aggregates in Alzheimer's disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD mutations and reveals that many variants beyond substitutions accelerate Aβ aggregation and are likely to be pathogenic. Truncations, substitutions, insertions, single- and internal multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are highly enriched in the polar N-terminal region of Aβ. This comparative atlas highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.© 2022. The Author(s).

JTD Keywords: amyloid-beta(1-42), determinants, disease, mutants, protein, secondary nucleation, Atomic-resolution structure

Yeast genetics made it possible to derive the first fundamental insights into prion composition, conformation, and propagation. Fast-forward 30 years and the same model organism is now proving an extremely powerful tool to comprehensively explore the impact of mutations in prion sequences on their function, toxicity, and physical properties. Here, we provide an overview of novel multiplexed strategies where deep mutagenesis is combined to a range of tailored selection assays in yeast, which are particularly amenable for investigating prions and prion-like sequences. By mimicking evolution in a flask, these multiplexed approaches are revealing mechanistic insights on the consequences of prion self-assembly, while also reporting on the structure prion sequences adopt in vivo.Copyright © 2022 Elsevier Ltd. All rights reserved.

JTD Keywords: aggregation, appearance, domains, inheritance, mutations, nucleation, physical basis, propagation, protein, Phase-separation

Plaques of the amyloid beta (A beta) peptide are a pathological hallmark of Alzheimer's disease (AD), the most common form of dementia. Mutations in A beta also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of A beta. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in A beta, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in A beta.

JTD Keywords: aggregation, kinetics, oligomers, onset, rates, state, Aggregation, Alzheimer disease, Alzheimer's, Amyloid, Amyloid beta-peptides, Computational biology, Deep mutagenesis, Dna mutational analysis, Genetics, Genomics, High-throughput nucleotide sequencing, Kinetics, Mutation, Nucleation, Oligomers, Onset, Plasmids, Precursor protein, Rates, S. cerevisiae, Saccharomyces cerevisiae, State, Systems biology

Blends with different ratios of polylactide and polyamide 6,10 (PA610) have been prepared by melt-mixing using a Brabender mixer equipment. Previously, a rheologically modified polylactide (PLAREx) was obtained through reactive extrusion using a multifunctional epoxide agent. It was expected that unreacted epoxy groups of PLAREx were able to improve the compatibility between the two polymers. SEM observations revealed a logical dependence of the morphology of immiscible phases with composition, and more interestingly a co-continuity at relatively low PA content (around 50%) was detected. This result contrasts with previous observations performed with non-modified PLA. Confined PA domains increased with the PA content and hardly crystallized at the typical crystallization temperature of the pure PA (195 °C). Synchrotron X-ray diffraction studies indicated that a PA crystallization at a lower temperature close to 120 °C was enhanced and led to a pseudohexagonal γ phase that differs from the characteristic layered structure of PA610. SAXS data revealed also that well differentiated lamellar entities could be assigned at both immiscible polymer phases. Clear differences were observed in the spherulitic morphologies attained under isothermal melt crystallization experiments. Results indicated that the texture of PLAREx spherulites was modified by the presence of PA. Compatibilization of PA molecules on the crystal lamellar boundaries of PLAREx led to an enhancement of the lamellar twisting frequency. Optical microscopy results also indicated that the crystal growth rate of PLAREx increased by the incorporation of PA, but in contrast this had an adverse effect on the nucleation process.

JTD Keywords: Crystal growth rate, Epoxy modified polylactide, Nucleation, Polyamide 6,10, Polyamide crystalline structure, Polyamide/polylactide blend morphology, Thermal properties