Proteomic Investigation of the Cellular Interactors of α-Synuclein Fibrils

α-Synuclein is an intrinsically disordered protein that is commonly expressed in neuronal cells where it is thought to play a role in neurotransmitter recycling. Aggregation of α-synuclein into amyloid fibrils has been linked to the development of a number of neurodegenerative diseases including Parkinson’s disease and dementia with Lewy bodies. Transmission of α-synuclein fibrils between neurons has been linked to the progression of α-synuclein associated diseases. Moreover, exposure of cells to α-synuclein fibrils has been shown to induce cellular dysfunction and ultimately cell death following internalisation. Therefore, in order to better understand the mechanisms by which α-synuclein fibrils disrupt cellular function, the identity of the protein interactors of α-synuclein fibrils, following cellular internalisation, is of considerable interest.
Herein, a system was developed by which α-synuclein fibrils can be assembled from recombinant monomer, internalised by a neuronal-like cell line, and isolated – both following internalisation and from cell lysate – using a magnetic biotin-streptavidin isolation system. Proteins that co-isolated with α-synuclein fibrils were then identified and separated from background binding proteins by quantitative mass spectrometry, to define the α-synuclein fibril interactome. Cellular pathways as well as protein complexes that were overrepresented in the α-synuclein fibril interactome were then characterised by a variety of bioinformatic techniques.
By this method a number of pathways were identified that may link α-synuclein fibril internalisation to cellular dysfunction. These included interference with the nucleo-cytoplasmic transport machinery, sequestration of translational proteins in- cluding entire ribosomal complexes, disruption of the cellular trafficking – particularly of the endoplasmic reticulum to the Golgi – and potentially inducing mitochondrial dysfunction. This work demonstrates the application of unbiased proteomic study to the characterisation of an α-synuclein fibril interactome, and highlights a number of avenues for future research.