摘要
目的 探讨细菌CsgA蛋白对α-突触核蛋白(α-synulcien,α-Syn)聚集的影响及CsgA蛋白促进帕金森病发生的可能机制。方法 在1.5 mg/mL的α-Syn单体蛋白中加入终浓度为0.01 mg/mL的CsgA蛋白R1结构域,采用硫黄素T染色法检测α-Syn的聚集;透射电镜分别观察R1,α-Syn和R1-α-Syn混合纤维的形态;分别将α-Syn纤维和R1-α-Syn混合纤维转导入稳定表达GFP标签的α-Syn的HEK293(Syn293)细胞中,观察对α-Syn聚集体形成的影响;分别用α-Syn纤维和R1-α-Syn混合纤维诱导Syn293细胞内形成聚集体,采用免疫荧光染色观察α-Syn聚集体是否具有路易小体的特征。结果 R1蛋白可以明显促进α-Syn的聚集,并且这种聚集体具有路易小体的特征。结论 细菌蛋白CsgA可促进α-Syn的聚集,这可能是细菌感染促进帕金森病发生的重要机制。
Objective To explore the effect of bacterial protein CsgA on the aggregation(α-Syn), and to illustrate the molecular mechanisms by which CsgA promote the onset of PD. Methods Experiment # 1: The aggregation of α-Syn(1.5 mg/ml) in the presence or absence of 0.01 mg/ml R1 as monitored by Thioflavin T fluorescence assay. Experiment # 2: The structure of the R1 pre-formed fibrils(PFFs), α-Syn PFFs, and R1-α-Syn mixed PFFs were observed by electron microscopy. Experiment # 3: α-Syn PFFs and R1-α-Syn mixed PFFs were transduced into HEK293 cells stably transfected with GFP-α-Syn(Syn293 cells). The formation of insoluble α-Syn aggregation was recorded. Experiment # 4: The characterization of intracellular aggregates induced by α-Syn PFFs and R1-α-Syn PFFs were investigated by immunofluorescence. Results R1 fragment of CsgA significantly promoted the aggregation of α-Syn. The R1-induced α-Syn PFFs show enhanced seeding activity in Syn293 cells. The aggregated indued by α-Syn PFFs and R1-α-Syn PFFs show characteristics of Lewy bodies. Conclusion R1, the key domain of CsgA, accelerates the aggregation of α-Syn. The CsgA-indued aggregation of α-Syn may play a role in the pathogenesis of Parkinson’s disease.
作者
孟兰霞
刘聪聪
张振涛
Men Lanxia;Liu Congcong;Zhang Zhentao(Departmental of Neurology,Renmin Hospital of Wuhan University,Wuhan 430060)
出处
《卒中与神经疾病》
2022年第5期401-404,409,共5页
Stroke and Nervous Diseases
基金
国家自然科学基金项目(81901090)。
