Global Center for Medical Engineering and Informatics, Osaka University, Osaka, Japan; Institute for Protein Research, Osaka University, Osaka, Japan.
Institute for Protein Research, Osaka University, Osaka, Japan.
J Biol Chem. 2021 Jan-Jun;296:100510. doi: 10.1016/j.jbc.2021.100510. Epub 2021 Mar 4.
Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged "KTK" segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-μM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.
多聚磷酸盐（polyPs）是一种存在于从细菌到哺乳动物等各种生物中的磷酸残基链，最近有报道称其可加速许多蛋白质的淀粉样纤维形成。然而，多聚磷酸盐如何促进这一过程尚不清楚。为了深入了解其机制，我们使用了各种物理化学方法来研究不同链长的多聚磷酸盐对超声依赖的α-突触核蛋白（α-syn）淀粉样形成的影响。尽管正磷酸盐和二磷酸盐表现出单一的最佳淀粉样形成浓度，但三磷酸盐和更长链的磷酸盐表现出两个最佳浓度，第二个最佳浓度低于正磷酸盐或二磷酸盐。随着多聚磷酸盐长度的增加，第二个最佳浓度显著降低。这表明，虽然较低多聚磷酸盐浓度下的最佳浓度是由带负电荷的磷酸基团与α-syn 的正电荷之间的相互作用引起的，但较高多聚磷酸盐浓度下的最佳浓度是由磷酸基团的 Hofmeister 盐析效应引起的，其中效应不依赖于净电荷。与四磷酸盐进行的 NMR 滴定实验与主成分分析相结合，表明在四磷酸盐的低浓度下，带负电荷的四磷酸盐与位于 N 端区域的四个 KTKEGV 重复中的带正电荷的“KTK”片段相互作用。在高浓度下，水合四磷酸盐会影响紧密的α-syn 暴露在表面的亲水性基团。总之，我们的结果表明，由 60 到 70 个磷酸组成的长链多聚磷酸盐在亚微摩尔浓度下诱导淀粉样形成，这与血液或组织中的多聚磷酸盐浓度相当。因此，这些发现可能确定了多聚磷酸盐在淀粉样相关疾病发病机制中的作用。
