Synphilin-1 inhibits alpha-synuclein degradation by the proteasome

Abstract

Intracellular deposits of aggregated alpha-synuclein are a hallmark of Parkinson's disease. Protein-protein interactions are critical in the regulation of cell proteostasis. Synphilin-1 interacts both in vitro and in vivo with alpha-synuclein promoting its aggregation. We report here that synphilin-1 specifically inhibits the degradation of alpha-synuclein wild-type and its missense mutants by the 20S proteasome due at least in part by the interaction of the ankyrin and coiled-coil domains of synphilin-1 (amino acids 331-555) with the N-terminal region (amino acids 1-60) of alpha-synuclein. Co-expression of synphilin-1 and alpha-synuclein wild-type in HeLa and N2A cells produces a specific increase in the half-life of alpha-synuclein, as degradation of unstable fluorescent reporters is not affected. Synphilin-1 inhibition can be relieved by co-expression of Siah-1 that targets synphilin-1 to degradation. Synphilin-1 inhibition of the proteasomal pathway of degradation of alpha-synuclein may help to understand the pathophysiological changes occurring in PD and other synucleinopathies.

Fig. 1

Time course of the degradation of alpha-synuclein by 20S proteasome. Purified alpha-synuclein (Snca) wild-type, A30P, E46K, and A53T were incubated with purified 20S proteasome as indicated in the figure. Results show representative Coomassie Blue-stained gels using 1 μg (3.57 μM) of the respective alpha-synuclein and 2.5 μg (173 nM) of 20S proteasome in the reaction mixtures

Fig. 2

Effect of synphilin-1 313–919 on the degradation of alpha-synuclein wild-type and its missense mutants by 20S proteasome. The panels show representative Coomassie Blue-stained gels of the degradation reactions performed in the absence (upper panels) and in the presence of synphilin-1 (Sncaip, lower panels) for alpha-synuclein (Snca) wild-type and the missense mutants (A30P, E46K, and A53T) as indicated. The quantifications for each of the reaction sets are shown in the corresponding graphical representations below each set of experiments. Reaction conditions were: 69.4 nM proteasome, 7 μM alpha-synuclein, and 0.75 μM synphilin-1 (331–919). Control reactions with synphilin-1and alpha-synuclein for 60 min in the absence of proteasome are also shown

Fig. 3

Effect of ankyrin and coiled-coil region of synphilin-1 on the degradation of alpha-synuclein by the 20S proteasome. The panels show representative Coomassie Blue-stained gels of the degradations reactions of alpha-synuclein (Snca) wild-type and its missense mutants by 20S proteasome in the presence of synphilin-1 (Sncaip, amino acids 331–555). Reaction conditions: 69.4 nM proteasome, 7 μM alpha-synuclein, and 0.92 μM synphilin-1 (331–515). Control reactions with synphilin-1 and alpha-synuclein for 60 min in the absence of proteasome are also shown

Fig. 4

Synphilin-1 inhibits the degradation of alpha-synuclein by interacting with the N-terminal region of alpha-synuclein. a Representative experiments analyzed by SDS-PAGE and Coomassie Blue staining and quantification of the degradation of N-terminal His-tag alpha-synuclein (His-Snca wt, 7 μM) full length by 20S proteasome (69.4 nM) in the absence and in the presence of synphilin-1 (Sncaip 331–919). b Similar experiments and quantification using N-terminal His-tag alpha-synuclein 1–50 (His-Snca 1–60, 7 μM)

Fig. 5

Dose-dependence of the synphilin-1 inhibition of alpha-synuclein degradation by the proteasome. Different amounts of synphilin-1 (Sncaip 313–919) were incubated with a fixed amount (3.57 μM) of the different types of alpha-synuclein (Snca wild-type, A30P, E46K, and A53T) and degradation was performed with 69.4 nM 20S proteasome. The amount of alpha-synuclein remaining after incubation of the reactions for 30–45 min was quantitated after analysis by SDS-PAGE and Coomassie Blue staining. Left graph shows the direct plot of % activity vs. synphilin-1 concentration in the assay, taking 100% as the activity obtained in the absence of synphilin-1. Right graph shows the log transformation of the direct data to calculate the IC_0.5, estimated to be 0.25 μM under this reaction conditions. Results are averaged from three different experiments with deviations below 10%

Fig. 6

Synphilin-1 inhibits the degradation of alpha-synuclein in HeLa and N2a cells. HeLa and N2a cells were transfected with alpha-synuclein (Snca wt) or co-transfected with alpha-synuclein and synphilin-1 (Sncaip). Cells 48 h after transfection were treated with cycloheximide (CHX) either in the absence or in the presence of 10 μM lactacystin for the times indicated. The panels show representative immunoblots with anti-alpha-synuclein antibodies, and anti-tubulin antibodies used as protein loading controls. Quantifications are shown in the right graphs as means ± SEM. from three different experiments. Upper panels, HeLa cells; lower panels, N2A cells

Fig. 7

Effect of synphilin-1 on the degradation of unstable fluorescent proteins. HeLa cells were transfected with EGFPu or EGFPd2 and co-transfected with synphilin-1 (Sncaip), as indicated. Cells 48 h after transfection were treated with cycloheximide (CHX). The panels show representative immunoblots with anti-EGFP antibodies, and anti-tubulin antibodies used as protein loading controls. Quantifications are shown in the right graph as means ± SEM from three different experiments

Fig. 8

Siah-1 effects on synphilin-1 and alpha-synuclein degradation. HeLa cells were single, double, or triple transfected with different plasmids. a Representative immunoblots of the steady-state expression of synphilin-1 (Sncaip) after transfection in the absence or in the presence of Siah-1, as indicated. The blots have been over-exposed in order to show synphilin-1 expression in the presence of transfected Siah-1. b HeLa cells 48 h after transfection with the indicated plasmids were treated with cycloheximide (CHX). Representative immunoblots with anti-Myc (synphilin-1, Sncaip) and anti-HA (Siah-1) are presented and data quantification are shown in d. c HeLa cells 48 h after transfection with the indicated plasmids were treated with CHX. Representative immunoblots with anti-alpha-synuclein (Snca) antibodies are presented and data quantification are shown in d. d Graph showing the quantification of data presented in b and c expressed as means ± SEM from three different experiments. Anti-tubulin antibodies were used as protein-loading controls