大鼠帕金森病模型中α-突触核蛋白对干扰素调节因子1的调控及其机制

Regulatory mechanism of interferon regulatory factor 1 byα-synuclein in mouse Parkinson's disease model

目的研究α-突触核蛋白(α-Syn)对干扰素调节因子1(IRF-1)表达的影响及其分子机制。方法选用α-Syn过表达的SHSY5Y细胞以及3月龄、6月龄A53Tα-Syn转基因小鼠,应用Real-time PCR和Western blot技术检测IRF-1表达的变化,免疫荧光染色和核浆蛋白分离技术观察IRF-1亚细胞定位情况。不同浓度的蛋白酶体抑制剂MG132(0、0.01、0.05、0.1、0.2、0.5、1、2μmol/L)以及溶酶体抑制剂氯喹(0、5、10、30、50、100、150、200μmol/L)处理SH-SY5Y细胞24 h,检测细胞存活率。选用不引起细胞损伤的最大浓度0.2μmol/LMG132和30μmol/L氯喹处理SH-SY5Y细胞24 h,检测IRF-1蛋白水平的变化。观察α-Syn对MDM2蛋白水平的影响,并进一步通过泛素化实验检测IRF-1泛素化水平的变化。结果α-Syn过表达不影响IRF-1 mRNA水平,却可显著上调其蛋白水平(P<0.01),并且IRF-1在胞核表达的比例升高,发生核移位现象(P<0.001)。不同浓度的MG132和氯喹处理后细胞存活率随浓度升高而逐渐降低,不引起细胞损伤的最大浓度分别为0.2μmol/L和30μmol/L。0.2μmol/L MG132处理后可加剧α-Syn引起的IRF-1蛋白水平的升高(P<0.01),而30μmol/L氯喹处理后IRF-1蛋白表达无明显变化。α-Syn过表达导致MDM2蛋白水平降低(P<0.01),进而抑制IRF-1的泛素化修饰。结论α-Syn通过泛素-蛋白酶体途径,抑制MDM2介导的IRF-1泛素化修饰,进而上调IRF-1蛋白表达。

Objective To investigate the molecular mechanism by whichα-synuclein(α-Syn)regulates interferon regulatory factor 1(IRF-1)expression.Methods SH-SY5Y cells overexpressingα-Syn and transgenic mouse model carrying humanα-Syn gene with A53T mutation(3 and 6 months old)were examined for IRF-1 mRNA and protein expressions using real-time PCR and Western blotting,respectively.The subcellular localization of IRF-1 was determined with immunofluorescence staining and cytoplasmic/nuclear protein isolation.The optimal concentrations of the proteasome inhibitor MG132(0.01-2.0μmol/L)and lysosomal inhibitor chloroquine(5-200μmol/L)for treatment of SH-SY5Y cells for 24 h were determined by examining the cell viability.SH-SY5Y cells were treated with 0.2μmol/L MG132 and 30μmol/L chloroquine for 24 h(the maximum dose that did not cause cell damage),and the changes of IRF-1 protein expressions was analyzed.The effects ofα-Syn on MDM2 protein expression and IRF-1 ubiquitylation were analyzed using Western blotting and ubiquitylation assay.Resultsα-Syn overexpression did not affect the mRNA level of IRF-1 but significantly increased its protein level(P<0.01).Inα-Synoverexpressing SH-SY5Y cells,IRF-1 translocation was observed from the cytoplasm to the nucleus(P<0.001).Treatment of the cells with 0.2μmol/L MG132 significantly aggravatedα-Syn-induced increase of IRF-1 protein expression(P<0.01)while 30μmol/L chloroquine produced no significant changes in IRF-1 level.α-Syn overexpression caused an obvious decrease of MDM2 protein level and further inhibited the ubiquitylation of IRF-1(P<0.01).Conclusionα-Syn blocks MDM2-mediated ubiquitylation of IRF-1 through ubiquitin proteasome pathway,thereby enhancing IRF-1 protein expression.