Institute of Molecular Biosciences, University of Graz, Graz, Austria.
PLoS One. 2010 Oct 27;5(10):e13700. doi: 10.1371/journal.pone.0013700.
Parkinson's disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive.
METHODOLOGY/PRINCIPAL FINDINGS: We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation.
CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.
帕金森病的特征是存在细胞质包涵体，称为路易体，其中包含聚集的α-突触核蛋白及其相互作用伙伴突触核蛋白 1。虽然已知突触核蛋白 1 在哺乳动物细胞中加速α-突触核蛋白的包涵体形成，但它对细胞毒性的影响仍不清楚。
方法/主要发现：我们在酵母酿酒酵母中单独或组合表达野生型突触核蛋白 1 或其 R621C 突变体，并监测蛋白质的细胞内定位和包涵体形成，以及对生长、氧化应激和细胞死亡的影响。我们发现野生型和突变型突触核蛋白 1 在酵母细胞中形成包涵体并加速α-突触核蛋白的包涵体形成，后者与丝氨酸 129 的磷酸化增强有关。突触核蛋白 1 包涵体与脂滴和内质网共定位。一致地，我们发现野生型和突变型突触核蛋白 1 与去污剂抗性膜域（称为脂筏）相互作用。突触核蛋白 1 的表达在指数培养物中不会引起明显的生长缺陷，这可能是由于形成聚集体和包涵体从子细胞逆行运输回母细胞。然而，当培养物接近静止期并在酵母细胞随后老化时，野生型和突变型突触核蛋白 1 均降低存活率并引发凋亡和坏死细胞死亡，尽管程度不同。最有趣的是，在缺乏 SIR2 （一种酵母中的 Sirtuin 蛋白）的老化细胞中，突触核蛋白 1 不会引发细胞毒性。这表明野生型细胞中突触核蛋白 1 的表达导致 SIR2 依赖性过程的失调，例如对营养饥饿的自噬流的维持。
结论/意义：我们的发现表明，野生型和突变型突触核蛋白 1 是与脂筏相互作用的蛋白质，当在酵母中表达时，它们形成包涵体并加速α-突触核蛋白的包涵体形成。突触核蛋白 1 从而诱导细胞毒性，野生型蛋白的作用最为明显，并且通过 SIR2 依赖性过程介导。
