Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation.

A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (alpha-Syn) upon differentiation with retinoic acid was used to study the impact of alpha-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced alpha-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. alpha-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating alpha-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 alpha/beta in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that alpha-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.