摘要
目的探讨NUS1突变体R290C互作蛋白组的变化及其与Lennox-Gastaut综合征(LGS)致病性的关系。方法构建野生型和突变型NUS1质粒并转染至人胚肾HEK293T细胞,48 h后采用Western blotting实验检测转染后HEK293T细胞NUS1蛋白的表达。采用免疫共沉淀、硝酸银染色、蛋白质谱-蛋白组学分析与野生型和突变型NUS1(R290C)发生互作的蛋白,鉴定出差异蛋白。采用基因本体(GO)、京都基因和基因组百科全书(KEGG)富集分析差异蛋白的分子功能和信号通路。利用DisGeNet数据库分析差异蛋白和疾病的关联。采用蛋白质-蛋白质互作(PPI)分析NUS1与蛋白折叠调控蛋白RTN4、DHDDS、发育性癫痫型脑病相关蛋白间的互作网络。结果(1)野生型、突变型NUS1质粒转染HEK293T细胞后NUS1蛋白表达的差异无统计学意义(t=0.536,P=0.620)。(2)与野生型NUS1质粒比较,突变型NUS1质粒转染后细胞中与NUS1互作的蛋白数量明显减少。蛋白质谱-蛋白组学分析共筛选出310个差异蛋白。(3)GO和KEGG富集分析显示差异蛋白主要参与蛋白折叠反应和翻译调控过程。(4)关联分析显示最相关的2类蛋白与额颞型痴呆和发育性癫痫性脑病相关。(5)PPI分析显示NUS1可能通过影响蛋白折叠信号通路,参与LGS等发育性癫痫性脑病的发生。结论NUS1突变体R290C改变其互作蛋白质谱系,可能通过调控蛋白折叠相关的信号,介导LGS等发育性癫痫性脑病的发生。
ObjectiveTo investigate the changes in interaction proteome of NUS1 mutant R290C and their relations with pathogenicity of Lennox Gastaut syndrome(LGS).MethodsThe wild-type and mutant NUS1(R290C)plasmids were constructed and transfected into human embryonic kidney HEK293T cells;48 h after that,NUS1 protein expression in HEK293T cells was detected by Western blotting.Co-immunoprecipitation,silver nitrate staining,and proteomic analysis were used to analyze the proteins interacted with wild-type or mutant NUS1 and identify the differential interacting proteins.Enrichment analyses of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were performed to annotate the molecular function and signaling pathways involved in the differential proteins.DisGeNet database was used to analyze the association between differential proteins and human diseases.Protein-protein interaction(PPI)was used to analyze the interaction network of NUS1 with protein folding regulatory proteins(RTN4 and DHDDS)and developmental epileptic encephalopathy related proteins.Results(1)There was no significant difference in NUS1 protein expression between the wild-type and mutant NUS1 transfected HEK293T cells(t=0.536,P=0.620).(2)Compared with that with wild-type NUS1 plasmid,number of proteins interacting with mutant NUS1 plasmid was significantly reduced in the transfected cells;310 differential interacting proteins were screened in the mutant NUS1.(3)GO and KEGG enrichment analyses showed that the differential proteins were mainly involved in protein folding reaction and translation regulation.(4)DisGeNet association analysis showed that the two most relevant proteins in the differential interacting proteins were associated with frontotemporal dementia and developmental epileptic encephalopathy.(5)PPI analysis showed that NUS1 may be involved in occurrence of neurological diseases such as LGS by affecting protein folding signaling pathways.Conclusion NUS1 mutant R290C alters its interacting protein lineage and mediates the development
作者
陈立志
石筱言
沈楠翔
范翠霞
叶子龙
李文斌
Chen Lizhi;Shi Xiaoyan;Shen Nanxiang;Fan Cuixia;Ye Zilong;Li Wenbin(Key Laboratory of Neuropathogenic Genes and Ion Channel Diseases in Ministry of Education,Institute of Neuroscience in Guangzhou Medical University,Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China;School of Medical Laboratory Technology,Changsha Medical University,Changsha 410219,China)
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2024年第11期1113-1119,共7页
Chinese Journal of Neuromedicine
基金
广东省基础与应用基础研究基金(2021A1515110792)
广州医科大学科研能力提升项目(2024SRP096)。
