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.

Accumulation of mutant proteins into misfolded species and aggregates is characteristic for diverse neurodegenerative diseases including the polyglutamine diseases. While several studies have suggested that polyglutamine protein aggregates impair the ubiquitin-proteasome system, the molecular mechanisms underlying the interaction between polyglutamine proteins and the proteasome have remained elusive. In this study, we use fluorescence live-cell imaging to demonstrate that the proteasome is sequestered irreversibly within aggregates of overexpressed N-terminal mutant Huntingtin fragment or simple polyglutamine expansion proteins. Moreover, by direct targeting of polyglutamine proteins for proteasomal degradation, we observe incomplete degradation of these substrates both in vitro and in vivo. Thus, our data reveal that intrinsic properties of the polyglutamine proteins prevent their efficient degradation and clearance. Additionally, fluorescence resonance energy transfer is detected between the proteasome and aggregated polyglutamine proteins indicative of a close and stable interaction. We propose that polyglutamine-containing proteins are kinetically trapped within proteasomes, which could explain their deleterious effects on cellular function over time.

The accumulation of aggregated ␣-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated ␣-synuclein binds to S6, a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitindependent. The IC 50 of aggregated ␣-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is ϳ1 nM. ␣-Synuclein has two close homologues, termed ␤-synuclein and ␥-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric ␣-and ␤-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric ␥-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC 50 of monomeric ␥-synuclein for the 20 S proteolysis was 400 nM. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although ␤-synuclein had no direct effect on proteasomal activity, co-incubating monomeric ␤-synuclein with aggregated ␣-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated ␣-synuclein when added before the aggregated ␣-synuclein. Co-incubating ␤-synuclein with ␥-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric ␥-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by ␤-synuclein resulted from binding to ␣-synuclein rather than binding to S6. Pull-down experiments demonstrated that recombinant monomeric ␤-synuclein does not interact with the proteasomal subunit S6, unlike ␣-synuclein, but ␤-synuclein does bind ␣-synuclein and competes with S6 for binding to ␣-synuclein. Based on these data, we hypothesize that the ␣-and ␥-synucleins regulate proteasomal function and that ␤-synuclein acts as a negative regulator of ␣-synuclein.