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

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.

α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast.

Parkinson's disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

DEAD-box RNA helicase Dbp4/DDX10 is an enhancer of α-synuclein toxicity and oligomerization.

Parkinson's disease is a neurodegenerative disorder associated with misfolding and aggregation of α-synuclein as a hallmark protein. Two yeast strain collections comprising conditional alleles of essential genes were screened for the ability of each allele to reduce or improve yeast growth upon α-synuclein expression. The resulting 98 novel modulators of α-synuclein toxicity clustered in several major categories including transcription, rRNA processing and ribosome biogenesis, RNA metabolism and protein degradation. Furthermore, expression of α-synuclein caused alterations in pre-rRNA transcript levels in yeast and in human cells. We identified the nucleolar DEAD-box helicase Dbp4 as a prominent modulator of α-synuclein toxicity. Downregulation of DBP4 rescued cells from α-synuclein toxicity, whereas overexpression led to a synthetic lethal phenotype. We discovered that α-synuclein interacts with Dbp4 or its human ortholog DDX10, sequesters the protein outside the nucleolus in yeast and in human cells, and stabilizes a fraction of α-synuclein oligomeric species. These findings provide a novel link between nucleolar processes and α-synuclein mediated toxicity with DDX10 emerging as a promising drug target.