Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Abstract

Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

“…By controlling the concentration of DNA priming the system, we can tune the final expression levels of proteins and coexpress proteins at controlled ratios. We have tested this combination on a variety of biological systems over the years, and demonstrated that the flexibility of cell-free protein expression is a great asset to study protein aggregation and prion-like fibrillation 15,[18][19][20][21] .…”

“…To evaluate the size of the oligomers and assemblies more precisely, we used Photon Counting Histograms (PCH) analysis to determine the stoichiometry of the complexes. We have described the PCH method in detail in previous publications 15,19,22 , and will provide a brief description here of the principle and analysis. To perform Photon Counting, the sample (expressed at the highest DNA loading) is diluted so that individual peaks can be fully resolved.…”

The RHIM within the M45 protein from murine cytomegalovirus forms heteromeric amyloid fibrils with RIPK1 and RIPK3

“…Fluorescence co-localisation experiments were applied to detect RHIM-based hetero-oligomers against a background of monomeric proteins and the potential competing formation of homo-oligomers 36,37 . In these experiments, multiple different RHIM-containing fusion proteins were mixed together and maintained in monomeric form in 8 M urea-containing buffer.…”

Mechanisms of strain diversity of disease-associated in-register parallel β-sheet amyloids and implications about prion strains

“…Another vigorously attempted biochemical approach for structural information of αSyn amyloids is assessment of cross-seeding efficiencies between αSyn molecules with familial-PD-associated mutations, e.g., between A30P and A53T [43]. Evaluation of aggregation formation and cross-reactions of in vitro-translated GFP-fusion mutant αSyn demonstrated that αSyn mutants with A53T, H50Q, or E46K spontaneously form large aggregates and efficiently co-aggregate with each other, whereas they do not co-aggregate with the wild-type or mutants with G51D or A30P; the latter group of three αSyn species formed smaller aggregates and co-aggregate each other within the group [44]. Those findings suggested existence of two mutually exclusive aggregation paths of αSyn amyloid formation.…”