基于质谱技术筛选酿酒酵母中Hrr25在自噬中的相互作用蛋白  

Screening of Hrr25-interacting Proteins in Autophagy in Saccharomyces cerevisiae Based on Mass Spectrometry

作　　者：姜文艳 徐佳钰 崔艳美 王娟[1] JIANG Wenyan;XU Jiayu;CUI Yanmei;WANG Juan(College of Environment and Life,Beijing University of Tecnology,Beijing 100124,China)

机构地区：[1]北京工业大学环境与生命学部,北京100124

出　　处：《河北师范大学学报（自然科学版）》2024年第1期77-84,共8页Journal of Hebei Normal University：Natural Science

基　　金：国家自然科学基金(31970044)。

摘　　要：Hrr25是酿酒酵母中具有丝氨酸/苏氨酸蛋白激酶活性的I型酪蛋白激酶家族成员.在自噬过程中Hrr25可以磷酸化多种蛋白调控自噬过程,然而目前对Hrr25的作用底物分析仍不明确,存在巨大的科研探究价值.Hrr25相互作用蛋白的鉴定可以通过免疫沉淀结合质谱分析来实现.构建了表达Hrr2513Myc蛋白的菌株,以Hrr2513Myc为靶蛋白进行免疫沉淀,将免疫沉淀的样品进行质谱检测.利用Venny2.1软件,得到在诱导自噬条件下与Hrr25特异性相互作用的蛋白.通过DAVID数据库进行基因本体论(gene ontology,GO)和京都基因和基因组百科全书(kyoto encyclopedia of genes and genome,KEGG)基因富集.结果在诱导自噬条件下筛选出321种特异与Hrr25相互作用的蛋白,根据P<0.05,GO富集共得到23项生物过程(biological process,BP)、16项细胞组成(cellular component,CC)、21项分子功能(molecular function,MF),KEGG富集分析到14条通路.通过免疫沉淀结合质谱技术,筛选出在自噬过程中与Hrr25互作的候选蛋白,为研究Hrr25与底物的相互作用及深入了解其调控自噬的分子机制提供新信息.Hrr25 is a member of the casein kinase 1 family with serine/threonine protein kinase activity in Saccharomyces cerevisiae.During autophagy,Hrr25 can phosphorylate a variety of proteins to regulate autophagy.However,the analysis of the substrates for Hrr25 is still unclear and has great scientific research value.Identification of Hrr25-interacting protein can be achieved by immunoprecipitation combined with mass spectrometry analysis.In this paper,a strain was constructed to express Hrr25-13Myc protein,and Hrr25-13Myc was used as the target protein for immunoprecipitation,and the immunoprecipitated samples were detected by mass spectrometry.Using Venny 2.1 software,proteins that specifically interact with Hrr25 under the condition of induced autophagy were obtained.Gene set enrichment analysis was performed by Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG)through DAVID database.Results showed that 321 proteins specifically interacting with Hrr25 were screened under the condition of induced autophagy.According to P<0.05,23 biological processes(BP),16 cellular compo-nents(CC),and 21 molecular functions(MF)were obtained by GO enrichment,while 14 pathways were obtained by K EGG enrichment.Through immunoprecipitation combined with mass spectrometry,candidate proteins interacting with Hrr25 during autophagy were screened,which provides new information for studying the interaction between Hrr25 and substrates and further understanding the molecular mechanism of its regulation of autophagy.

关 键 词：Hrr25 自噬 酿酒酵母 质谱 

分 类 号：Q25[生物学—细胞生物学]