Kong Li-Xiu, Zeng Cheng-Ming
Shaanxi Normal University, School of Chemistry and Chemical Engineering, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Xi'an, 710119, China.
Biochemistry (Mosc). 2017 Feb;82(2):156-167. doi: 10.1134/S0006297917020079.
Preformed amyloid fibrils can act as seeds for accelerating protein fibrillation. In the present study, we examined the effects of preformed seeds on lysozyme amyloid fibrillation in the presence of two distinct inhibitors - epigallocatechin (EGC) and polyethylene glycol 2000 (PEG). The results demonstrated that the effects of fibrillar seeds on the acceleration of lysozyme fibrillation depended on the aggregation pathway directed by an inhibitor. EGC inhibited lysozyme fibrillation and modified the peptide chains with quinone moieties in a concentration-dependent manner. The resulting aggregates showed amorphous off-pathway morphology. Preformed fibril seeds did not promote lysozyme fibrillation in the presence of EGC. PEG also inhibited lysozyme fibrillation, and the resulting aggregates showed on-pathway protofibrillar morphology. In contrast, the addition of fibril seeds into the mixture of lysozyme and PEG significantly stimulated fibril growth. Assays of cell viability showed that both EGC and PEG inhibited the formation of cytotoxic species. In accordance with thioflavine T data, the seeds failed to alter the cell-damaging potency of the EGC-directed off-pathway aggregates, but increased the cytotoxicity of the PEG-directed on-pathway fibrils. We suggest that the pattern of interaction between lysozyme and an inhibitor determines the pathway of aggregation and therefore the effects of seeding on amyloid formation. EGC covalently modified lysozyme chains with quinones, directing the aggregation to proceed through an off-pathway, whereas PEG affected the protein in a noncovalent manner, and fibril growth could be stimulated under seeding through an on-pathway.
预先形成的淀粉样纤维可以作为加速蛋白质纤维化的种子。在本研究中，我们研究了在两种不同的抑制剂——表没食子儿茶素（EGC）和聚乙二醇2000（PEG）存在的情况下，预先形成的种子对溶菌酶淀粉样纤维化的影响。结果表明，纤维状种子对溶菌酶纤维化加速的影响取决于抑制剂所引导的聚集途径。EGC抑制溶菌酶纤维化，并以浓度依赖的方式用醌基团修饰肽链。所形成的聚集体呈现无定形的非途径形态。在EGC存在的情况下，预先形成的纤维种子不会促进溶菌酶纤维化。PEG也抑制溶菌酶纤维化，所形成的聚集体呈现途径上的原纤维形态。相反，将纤维种子添加到溶菌酶和PEG的混合物中会显著刺激纤维生长。细胞活力测定表明，EGC和PEG都抑制细胞毒性物质的形成。与硫黄素T数据一致，种子未能改变EGC引导的非途径聚集体的细胞损伤能力，但增加了PEG引导的途径上的纤维的细胞毒性。我们认为，溶菌酶与抑制剂之间的相互作用模式决定了聚集途径，因此也决定了种子对淀粉样蛋白形成的影响。EGC用醌共价修饰溶菌酶链，并引导聚集通过非途径进行，而PEG以非共价方式影响蛋白质，并且在种子存在的情况下，通过途径上的方式可以刺激纤维生长。
