Crk1/2与CrkL缺失导致足细胞损伤的蛋白质组学分析

Proteomics analysis in Crk1/2 and CrkL knockdown podocyte

目的探讨Crk1/2与CrkL缺失导致足细胞损伤过程中胞内蛋白表达变化情况。方法利用小干扰RNA转染方法对足细胞内Crk1/2与CrkL进行单敲降及双敲降;通过非标记液相色谱串联质谱方法对Crk1/2与CrkL单敲降及双敲降的足细胞进行蛋白质组学分析;对得到的差异蛋白进行基因本体(GO)分析和京都基因和基因组百科全书(KEGG)分析;采用Western blot试验对差异蛋白进行验证。结果Crk1/2与CrkL单敲降及双敲降的足细胞与正常足细胞比较,共发现98个差异蛋白,GO分析和KEGG分析提示多数上调蛋白富集在代谢途径,多数下调蛋白富集在信号转导途径,如抑制和激活蛋白1、磷脂酰肌醇三激酶-丝氨酸/苏氨酸激酶和环磷酸腺苷信号途径。经Western blot试验验证,细胞代谢相关蛋白超氧化物歧化酶2(SOD2)表达上调,信号转导相关蛋白LRP1表达上调、c-Jun表达下调,细胞骨架损伤相关蛋白TPM4表达上调、Cdc42EP1表达下调。结论Crk1/2与CrkL缺失可通过调节代谢途径及相关信号转导途径导致足细胞损伤,TPM4、SOD2、LRP1、c-Jun和Cdc42EP1参与损伤过程,具体分子机制值得进一步探讨。

Objective To explore the changes of intracellular protein during podocyte injury caused by the absence of Crk1/2 and CrkL.Methods Specific knockdown of Crk1/2,CrkL and both by transfected with indicated small interfering RNA.Label-free liquid chromatography tandem mass spectrometry method was used to compare the protein profiles in Crk1/2,CrkL or both knockdown podocytes and normal podocytes.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to characterize the properties of the differentially expressed proteins.Western blot was used to verify the differentially expressed proteins.Results A total 98 proteins were identified to be significantly altered in Crk1/2,CrkL and both knockdown podocytes.GO analysis and KEGG analysis revealed that most of upregulated proteins were involved in metabolic process and most of downregulated proteins were involved in signaling transduction pathway such as Rap1,PI3K-Akt and cAMP signaling pathway.According to Western blot verification,cell metabolism-related protein SOD2 was up-regulated,signal transduction-related protein LRP1 was up-regulated,while c-Jun was down-regulated,cytoskeletal-related protein TPM4 was up-regulated,while Cdc42EP1 was down-regulated.Conclusion The loss of Crk1/2 and CrkL may cause podocyte injury by regulating metabolic process and related signal transduction pathway.TPM4,SOD2,LRP1,c-Jun and Cdc42EP1 were involved in the damage process and the specific molecular mechanisms were worth further discussion.