PDI抑制α-synuclein纤维化聚集作用机制研究

Inhibition Mechanisms of Protein Disulfide Isomerase on α-Synuclein Fibril Aggregation

天然无结构蛋白?-synuclein在帕金森症(PD)患者脑部的路易小体中异常聚集,被认为是引起PD的重要原因之一,但是目前关于?-synuclein的聚集机制仍没有定论.蛋白质二硫键异构酶(PDI)是细胞内质网中重要的分子伴侣蛋白,能够阻止内质网中无结构蛋白的聚集.在PD患者的脑细胞内发现PDI过量表达,且酶活性位点半胱氨酸被亚硝基化使其活性受到抑制.体外实验证明,PDI能够抑制?-synuclein的聚集,但其具体的分子机制还不清楚,研究PDI抑制?-synuclein聚集的具体机制可能对于PD治疗有重要意义.该文利用核磁共振(NMR)方法研究了?-synuclein与PDI的相互作用,发现?-synuclein与PDI的结合位点位于?-synuclein的N端;将PDI所有的6个半胱氨酸突变成丝氨酸,得到突变体PDI C-S,发现?-synuclein与PDI C-S的结合位点则位于其C末端;荧光实验结果表明突变体PDI C-S对?-synuclein纤维化聚集的抑制作用减弱,说明PDI抑制?-synuclein的纤维化聚集主要是通过与?-synuclein的N端残基结合来实现的.

Abnormal aggregation of?-synuclein,an intrinsically disordered protein in Lewy bodies,is considered a hallmark of Parkinson's disease(PD).The mechanisms underlying abnormal-synuclein aggregation,however,are yet to be understood.Protein disulfide isomerase(PDI)is an important molecular chaperone in the endoplasmic reticulum,which can prevent disordered protein forming aggregation.PDI is shown to be overexpressed in the brain of PD patients,but with reduced activity due to S-nitrosylation of the cysteine residues in the active site.In vitro experiments have demonstrated that PDI can inhibit the aggregation of?-synuclein with unknown mechanisms.Hence,it may be of importance to study the inhibition mechanisms of PDI on?-synuclein aggregation.In this study,the interaction between?-synuclein and PDI was studied by NMR spectroscopy.It was found that the binding sites between the two are located on the N-terminal of?-synuclein.A variant PDI(PDI C-S)was prepared,in which all six cysteines were mutated to serines.It was found that thebinding sites between PDI C-S and?-synuclein are located on the C-terminus of?-synuclein.The results of thioflavin T(ThT)fluorescence experiment showed that the inhibition activity of PDI C-S on?-synuclein aggregation was lower than the wild type,suggesting that wild type PDI may inhibit?-synuclein aggregation through binding to its N-terminal.