Samuel Filsy, Flavin William P, Iqbal Sobia, Pacelli Consiglia, Sri Renganathan Sri Dushyaanthan, Trudeau Louis-Eric, Campbell Edward M, Fraser Paul E, Tandon Anurag
From the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada.
the Integrative Cell Biology Program.
J Biol Chem. 2016 Feb 26;291(9):4374-85. doi: 10.1074/jbc.M115.705095. Epub 2015 Dec 30.
Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.
虽然在正常大脑中可检测到痕量水平的磷酸化α-突触核蛋白（α-syn），但在帕金森病大脑的路易小体中积累的几乎所有α-syn在丝氨酸129（Ser-129）处都发生了磷酸化。磷酸丝氨酸残基的作用及其对α-syn结构、功能和细胞内积累的影响尚不清楚。在这里，α-syn与靶向Ser-129的激酶polo样激酶2（PLK2）共表达，用于生成磷酸化α-syn以进行生物物理和生物学特性分析。尽管磷酸化α-syn异构体的原纤维对磷酸酶和蛋白酶仍敏感，但可更早检测到其错误折叠和原纤维形成。α-syn单体与膜的结合因磷酸化而受到不同影响，这取决于与帕金森病相关的突变。野生型α-syn与突触前膜的结合不受磷酸化影响，而A30P α-syn的结合大大增加，A53T α-syn的结合略有降低，这表明羧基末端对氨基末端膜结合有远端影响。内吞小泡介导的预形成原纤维内化到非神经元细胞和多巴胺能神经元中的效率与α-syn膜结合效率相匹配。最后，磷酸化α-syn异构体增强了内化小泡膜的破坏，这是错误折叠的细胞外或腔内α-syn进入胞质α-syn的一种潜在方式。我们的结果表明，即使在低水平的α-syn内化时，小泡通透性的阈值也很明显，这与减少α-syn错误折叠细胞间传播的治疗策略相关。
