The accumulation of aggregated ␣-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how ␣-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated ␣-synuclein strongly inhibited the function of the 26 S proteasome. The IC 50 of aggregated ␣-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nM. Aggregated ␣-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nM. In contrast, the IC 50 of aggregated ␣-synuclein for 20 S proteasomal activity was > 1 M. This suggests that aggregated ␣-synuclein selectively interacts with the 19 S cap. Monomeric ␣-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric ␣-synuclein inhibited the activity of the 20 S proteasomal core with an IC 50 > 10 M, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 M, and exhibited only partial inhibition (50%) of the 26 S ubiquitinindependent proteasomal activity at doses up to 10 mM. Binding studies demonstrate that both aggregated and monomeric ␣-synuclein selectively bind to the proteasomal protein S6, a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated ␣-synuclein could be mediated by interaction with S6.

Parkin and alpha-synuclein are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Parkin is present in Lewy bodies and axonal spheroids in brains affected by PD, and mutations in parkin cause hereditary forms of Parkinsonism. Alpha-synuclein is a major component of Lewy bodies and is associated with rare cases of PD. We now show that parkin binds to alpha-synuclein, including conditions associated with alpha-synuclein aggregation. Parkin and alpha-synuclein complexes were observed in BE-M17 cells under basal conditions, in BE- M17 cells under oxidative conditions and in brains from control or PD donors. Double staining of PD brains shows parkin and alpha-synuclein co-localize to the same pathological structures (both Lewy bodies and axonal spheroids). These results suggest that parkin interacts with alpha-synuclein and could contribute to the pathophysiology of PD more generally than was previously considered.