南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (11): 1641-1648.doi: 10.12122/j.issn.1673-4254.2021.11.07

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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

牟斐斐，陈 曦，杜希恂，焦 倩，毕明霞，姜 宏   

  1. 青岛大学国家生理学重点（培育）学科，山东 青岛 266071
  • 出版日期:2021-11-20 发布日期:2021-12-10

Regulatory mechanism of interferon regulatory factor 1 by α-synuclein in Parkinson's disease models

MU Feifei, CHEN Xi, DU Xixun, JIAO Qian, BI Mingxia, JIANG Hong   

  1. State Key Disciplines of Physiology (Incubation), Department of Physiology, Qingdao University, Qingdao 266071, China
  • Online:2021-11-20 Published:2021-12-10

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摘要： 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SH-SY5Y细胞以及3月龄、6月龄A53T α-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、2 μmol/L）以及溶酶体抑制剂氯喹（0、5、10、30、50、100、150、200 μmol/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0.2 μmol/L MG132和30 μmol/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-1mRNA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0.2 μmol/L和30 μmol/L。0.2 μmol/LMG132处理后可加剧α-Syn引起的IRF-1蛋白水平的升高（P<0.01），而30 μmol/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。

关键词: 帕金森病, α-突触核蛋白, 干扰素调节因子1, 神经免疫

Abstract: Objective To investigate the molecular mechanism by which α-synuclein (α-Syn) regulates interferon regulatory factor 1 (IRF-1) expression. Methods SH-SY5Y cells overexpressing α-Syn and transgenic mouse model carrying human α-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 (0.01-2.0 μmol/L) and lysosomal inhibitor chloroquine (5-200 μmol/L) for treatment of SH-SY5Y cells for 24 h were determined by examining the cell viability. SH-SY5Y cells were treated with 0.2 μmol/L MG132 and 30 μmol/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 of α-Syn on MDM2 protein expression and IRF-1 ubiquitylation were analyzed using Western blotting and ubiquitylation assay. Results α-Syn overexpression did not affect the mRNA level of IRF-1 but significantly increased its protein level (P<0.01). In α-Syn-overexpressing SH-SY5Y cells, IRF-1 translocation was observed from the cytoplasm to the nucleus (P<0.001). Treatment of the cells with 0.2 μmol/L MG132 significantly aggravated α-Syn-induced increase of IRF-1 protein expression (P<0.01) while 30 μmol/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). Conclusion α-Syn blocks MDM2-mediated ubiquitylation of IRF-1 through ubiquitin proteasome pathway, thereby enhancing IRF-1 protein expression.

Key words: Parkinson's disease, α-synuclein, interferon regulatory factor 1, neuroimmune