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.

Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with diverse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.

Cellular protein quality control comprises a network of chaperones that maintain the proteome viability by performing key cellular tasks such as degrading or remodeling misfolded proteins. Bacterial Caseinolytic proteases (Clp) which are responsible for protein degradation include powerful ring-shaped AAAþ (ATPases Associated with diverse cellular Activities) motors with a central narrow pore that unfold and translocate tagged abnormal proteins. Clp ATPase machines thread substrate proteins (SPs) through their central channel by using repetitive ATP-driven subunit motions coupled with axial mechanical forces exerted onto the SP. Here we perform multiscale molecular simulations of ClpYDI and Titin I27 to mimic and contrast laser optical tweezer (LOT) experiments, in which the SP N-terminus is restrained, with in vivo ClpY-mediated unfolding and translocation, in which the SP is not restrained at the N-terminus. This allows us to shed light on the effects of restraining forces and SP mechanical direction probed on Clp-mediated unfolding mechanism. The external LOT restraint limits ClpY-mediated pulling along the N-C direction of the SP, which yields unfolding of I27 via a shearing mechanism. By contrast, in vivo-like ClpY-action results in pulling along softer mechanical directions and I27 is unfolded via an unzipping mechanism. We find that factors that affect these distinct mechanisms are SP-ClpY surface interactions, the size of the SP relative to the ClpY pore size, the SP mechanical resistance, and the presence of other substrate domains.