Inhibition of 26S proteasome activity by α‐synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1

Abstract Parkinson's disease (PD) is characterized by aggregation of α‐synuclein (α‐syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α‐syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α‐syn protein levels and the impact of α‐syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α‐syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α‐syn and S129A variant on the abundance and stability of most yeast proteins using a genome‐wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α‐syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α‐syn. Elevated levels of Rpn14 enhanced the growth inhibition by α‐syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α‐syn and stabilizes pS129 α‐syn. The expression of α‐syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α‐syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α‐syn‐mediated proteasome dysfunction.

R1/R2 > 1 (decreased protein stability) R1/R2 < 1 (increased protein stability) Figure S1.Fluorescence intensity ratios from flow cytometry data support the results obtained in tFT-screen.Flow cytometry was conducted with selected tFT-strains expressing αSyn, S129A or with empty vector control.Intensity of fluorescence signal derived from mCherry and sfGFP was measured for 10000 single cells and mCherry/sfGFP ratios calculated for control, αSyn or S129A expressing cells.R1/R2 indicates ratio mCherry/GFP of control cells (R1) to ratio mCherry/GFP of αSyn or S129A expressing cells (R2) as log2.Significance of differences was calculated with t-test versus control cells (*p < 0.05; **p < 0.01; ***; p < 0.001, n=3).**  (a) Immunoblot analysis of Tet-RPT4 strain expressing GPD-driven RPN14 from CEN plasmid, GAL1-driven αSyn-GFP or S129A-GFP.Empty vector (EV) was used as control.Yeast cells were grown overnight in a galactose-containing medium to induce αSyn expression.The Tet promoter was downregulated by simultaneous addition of 10 μg/mL doxycycline (Dox) to the growth medium.(+) indicates Tet-ON, and (-) indicates Tet-OFF.Immunoblotting analysis was performed with anti-ubiquitin, αSyn or pS129 antibodies.GAPDH was used as loading control.(b) Densitometric analysis of the immunodetection of the ubiquitin conjugates in Tet-RPT4 strain relative to GAPDH.The significance of differences was calculated with a t-test relative to EV control (*, p < 0.05; **, p < 0.01).(c) Densitometric analysis of αSyn protein levels from Tet-RPT4 (A) relative to the GAPDH loading control.(d) Densitometric analysis of pS129 fraction relative to αSyn signal.(e) Immunoblot analysis of Tet-RPT4 Δrpn14 strain expressing αSyn-GFP, S129A-GFP, RPN14 or empty vector (EV) as control, performed as in (a).(f) Densitometric analysis of the immunodetection of the ubiquitin conjugates in Tet-RPT4 Δrpn14 strain relative to GAPDH.The significance of differences was calculated with t-test relative to EV control (**, p < 0.01).(g) Densitometric analysis of αSyn protein levels from (e) relative to GAPDH.(h) Densitometric analysis of pS129 fraction relative to αSyn signal in Tet-RPT4 Δrpn14 strain.

Figure S2 .Figure S3 .Figure S4 .
Figure S2.Spatial Analysis of Functional Enrichment (SAFE) (http://www.thecellmap.org)analysis of the identified proteins with significantly changed stability in presence of αSyn or S129A, compared to empty vector control.Specific biological processes that are enriched upon αSyn expression (green) or S129A expression (blue) are depicted on the network map.αSyn expression revealed enrichment of proteins involved in DNA replication & repair, mitosis, mRNA processing, nuclear transport, transcription and mitochondria.In presence of S129A the hits are functionally enriched mainly in the categories of DNA replication & repair and glycosylation & protein folding.

Figure S6 .
Figure S6.Native expression of Rpn14 increases αSyn toxicity.Growth assays of yeast cells, expressing αSyn, S129A or S129D in wild type, Δrpn14 or Δnas6 strains at 30 o C (a) or 37 o C (b). (c) Propidium iodide (PI) fluorescence intensity and side scatter (SSC) of cells assessed with flow cytometry analysis.Representative flow cytometry charts illustrating the sub-populations of yeast cells with higher fluorescent intensities (P1) than the background.Cells expressing different αSyn variants or empty vector (EV) control after 6 h induction of expression at 30 o C (c) or at 37 o C (d) were stained with 12.5 μg/ml PI for 30 min.The percentage of PI positive cells at 30 o C (e) or at 37 o C (f) is presented.A total of 10000 cells per experiment were counted.Significance of differences was calculated with one-way ANOVA with Newman-Keuls post-hoc test (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; n = 3).

Figure S8 .Figure S9 .
Figure S8.Expression of S129D increases the accumulation of ubiquitinated proteins.(a) Immunoblot analysis of Tet-RPT2 strain expressing GPD-driven RPN14, GAL1-driven αSyn-GFP or S129D-GFP.Yeast cells were grown overnight in galactose-containing medium to induce αSyn expression.The Tet promoter was repressed by simultaneous addition of 10 μg/ml doxycycline to the growth medium.(+) indicates Tet-ON, and (-) Tet-OFF.Immunoblotting analyses were performed with anti-ubiquitin or αSyn antibodies.GAPDH was used as a loading control.(b) Densitometric analysis of the immunodetection of the ubiquitin conjugates in Tet-RPT2 strain relative to GAPDH.Significance of differences was calculated with ttest relative to the corresponding αSyn +/-Rpn14 (*, p < 0.05; **, p < 0.01).

Figure S10 .
Figure S10.Expression of αSyn or elevated level of Rpn14 leads to accumulation of ubiquitinated proteins upon downregulation of Tet-RPT6.(a)Immunoblot analysis of Tet-RPT6 strain expressing GPD-driven RPN14 from CEN plasmid, GAL1-driven αSyn-GFP or S129A-GFP.Empty vector (EV) was used as control.Yeast cells were grown overnight in a galactose-containing medium to induce αSyn expression.The Tet promoter was downregulated by simultaneous addition of 10 μg/mL doxycycline (Dox) to the growth medium.(+) indicates Tet-ON; (-) Tet-OFF.Immunoblotting analysis was performed with ubiquitin, αSyn or pS129 antibodies.GAPDH was used as a loading control.(b) Densitometric analysis of the immunodetection of the ubiquitin conjugates in Tet-RPT6 strain relative to GAPDH.(c) Densitometric analysis of αSyn protein levels from Tet-RPT6 (a) relative to the GAPDH loading control.(d) Densitometric analysis of pS129 fraction relative to αSyn signal.The significance of differences was calculated with t-test relative to EV control (*, p < 0.05).(e) Immunoblot analysis of Tet-RPT6 Δrpn14 strain expressing αSyn-GFP, S129A-GFP, RPN14 or empty vector (EV) as control, performed as in (a).(f) Densitometric analysis of the immunodetection of the ubiquitin conjugates in Tet-RPT6 Δrpn14 strain relative to GAPDH.(g) Densitometric analysis of αSyn protein levels from (e) relative to GAPDH.(h) Densitometric analysis of pS129 fraction relative to αSyn signal in Tet-RPT6 Δrpn14 strain.

Figure S11 .
Figure S11.Proteasome activity assays.(a) Wild type (WT) yeast cells expressing GPD-driven RPN14 from CEN plasmid, GAL1-driven αSyn or S129A from 2μ plasmid, or empty vector (EV) as control were collected after 6 hours of GAL1 induction.The 26S proteasomal activity in crude protein extracts was monitored by measuring the hydrolysis of the fluorogenic peptide Suc-LLVY-AMC by detecting relative fluorescence units (RFU).(b) 26S proteasomal activity in Δrpn14 strain, performed similarly as in (a).(c) Crude protein extracts from wild type cells or Δrpn14 cells (d) were preincubated with 100 μM proteasome inhibitor MG132 for 10 min prior to measurement as control.Lack of proteasome activity is indicative for the specificity of the assay.(e) HEK cells were transfected with constructs expressing PAAF1, αSyn or S129A under the control of the CMV promoter.EV -empty vector.The 26S proteasomal activity was assayed in crude protein extracts by measuring the hydrolysis of Suc-LLVY-AMC.(f) HEK protein extracts were preincubated with 100 μM proteasome inhibitor MG132 for 10 min prior to the measurement presented in (e).