Sumoylation Protects Against β-Synuclein Toxicity in Yeast.

Blagovesta Popova,Jasmin Mischke,Patricia Arendarski,Alexandra Kleinknecht,Gerhard H Braus,Dan Wang

doi:10.3389/fnmol.2018.00094

Abstract

Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson’s disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, β and γ isoforms. β-synuclein (βSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of βSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and βSyn exacerbates the cytotoxicity. This suggests an important role of βSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and βSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing βSyn. This corroborates a protective role of the cellular sumoylation machinery against βSyn-induced toxicity. Upregulation of sumoylation significantly reduced βSyn aggregate formation. This is an indirect molecular process, which is not directly linked to βSyn sumoylation because amino acid substitutions in the lysine residues required for βSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for βSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of βSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect βSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular βSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between βSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to βSyn on eukaryotic cells.

Highlights

Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra in the midbrain and formation of intracellular protein inclusions called Lewy bodies (Kalia and Lang, 2015)
We showed that SUMO is an important modulator of protein stability. βSyn is sumoylated in yeast at three lysine residues and sumoylation supports aggregate formation
We demonstrate in yeast cells that intact sumoylation machinery is prerequisite for the degradation of soluble as well as aggregated βSyn protein. βSyn is degraded mainly by the 26S proteasome, in contrast to αSyn that is degraded predominantly by the autophagy/vacuolar pathway

Summary

Introduction

Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra in the midbrain and formation of intracellular protein inclusions called Lewy bodies (Kalia and Lang, 2015). One of the major components of Lewy bodies is the small and highly abundant protein α-synuclein (αSyn; Spillantini et al, 1998). ΑSyn is part of a family of proteins that includes β-synuclein (βSyn) and γ-synuclein (γSyn). ΑSyn and βSyn are localized predominantly at presynaptic nerve terminals, whereas γSyn is abundant in the peripheral nervous system (Li et al, 2002; Mori et al, 2002). The most notable difference between the two proteins is that βSyn lacks 11 residues in the central most hydrophobic region, referred to as non-amyloid-β component (NAC) region, involved in fibril formation and aggregation. All three synucleins are intrinsically unstructured when isolated under physiological conditions and adopt helical structures in their N-terminal domains upon binding to lipid vesicles (Sung and Eliezer, 2006; Ducas and Rhoades, 2012)

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