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.
Highlights
Thought to arise from the formation of small soluble oligomers[10,11], which form early in the aggregation pathway, rather than from the accumulation of mature fibrils
WT α-synuclein and 5 disease-associated α-synuclein mutants were expressed in Leishmania Tarentolae Extracts (LTE), with fluorescent proteins fused to their C-termini
The samples were observed after 2 h of expression at 27 °C, as the saturation of overall fluorescence signals the end of active protein production in LTE
Summary
Thought to arise from the formation of small soluble oligomers[10,11], which form early in the aggregation pathway, rather than from the accumulation of mature fibrils. The proteins of interest have to be rescued from insoluble fractions by forcing the protein into a non-oligomeric state using chemical or thermal denaturation After this delicate step, one has to create in vitro conditions that would mimic their physiological aggregation. Since the self-assembly can proceed through multiple steps of dimerization, oligomerization and nucleation of fibrils, the ability to detect early and rare events in the kinetic process becomes crucial[27] In this context, single-molecule fluorescence spectroscopy is especially attractive as they allow the detection of sub-populations in heterogeneous samples[27,28]. The use of genetically encoded fluorophores enabled a direct observation of the expressed proteins without purification, denaturation or labelling steps This combination of methods enabled to quantify concentration- and temperature-dependent aggregation, as well as co-aggregation between proteins tagged with different fluorophores. Applied to wild-type (WT) α-synuclein and five of its naturally occurring and pathogenic mutants (A30P7,31, E46K32, H50Q33,34, G51D35,36 and A53T37), the toolbox revealed an unexpected segregation of α-synuclein mutants into two different behaviors
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