The misfolding and aggregation of soluble proteins into insoluble amyloid deposits is associated with a range of neurodegenerative diseases. Amyloid deposits in these diseases are generally primarily composed of a single disease-specific protein, and the main pathological feature of Parkinson’s disease is the presence of alpha-synuclein positive cytoplasmic inclusions termed Lewy bodies.
The transition from soluble monomeric to fibrillar alpha-synuclein involves the population of multiple intermediate species, including oligomeric and protofibrillar structures. These species have differential toxicities, with certain oligomeric states now appearing to be the major toxic species. While alpha-synuclein is the major component of Lewy bodies, a range of other molecules are also associated with these deposits in vivo including molecular chaperones and other proteins involved in cellular quality control processes, as well as many proteins with a high intrinsic aggregation propensity.
We couple cell-free expression with single molecule techniques to rapidly screen Lewy body components and alpha-synuclein interactors for their ability to bind to each of these individual species. This approach has allowed the direct comparison of the affinity of on the order of 100 potential alpha-synuclein interactors to multiple species along the aggregation pathway. This screen has uncovered a number of potent interactors to specific species of alpha-synuclein, and characterisation of these interactions has revealed insights into how these proteins influence specific microscopic steps in amyloid formation.