Hu Mingyang, Fang Jiuyuan, Wang Huijuan, Zhou Sijie
The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Zhengzhou University School of Medical Sciences, Zhengzhou, China.
Front Genet. 2021 Apr 7;12:657140. doi: 10.3389/fgene.2021.657140. eCollection 2021.
Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is complicated. A thorough understanding of GSK-3β is crucial to understand the mechanism of diabetic nephropathy. To analyze the roles of GSK-3β in podocytes, GSK-3β knockdown lentivirus by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9 was applied to establish stable cell lines. Mass spectrometry was utilized to search for differentially expressed proteins. Consequently, we found 34 proteins with higher levels and 115 proteins with lower levels in GSk-3β knockdown cells than in control cells and identified 581 phosphosites with higher phosphorylation levels and 288 phosphosites with lower phosphorylation levels. We performed functional enrichment analysis of these proteins and phosphorylated proteins based on public databases. Enrichment analysis revealed that GSK-3β participates in the spliceosome, Hippo signaling pathway, actin binding, structural molecule activity, and other pathways. Then, we used motif analysis of phosphate sites to determine 89 conserved motifs based on 1,068 phosphoserine (pS) sites and 15 conserved motifs in view of 104 phosphothreonine (pT) sites. Additionally, protein-protein interaction network analysis was carried out using the STRING database. Cytoscape's add-on Molecular Complex Detection (MCODE) was used to analyze key and core protein groups. In quantitative differential protein analysis, four MCODEs were obtained, and 22 MCODEs were obtained in the analysis of the phosphoproteome of differentially expressed proteins. Finally, we analyzed the kinase regulatory network in podocytes after GSK-3β knockdown and identified 299 protein kinases and 3,460 significantly changed phosphorylation modification sites on 1,574 proteins. These results will be valuable for further research on GSK-3β.
糖尿病肾病是全球最常见的慢性肾脏病，也是终末期肾病（ESRD）的主要病因。足细胞的结构完整性对于肾小球的正常功能至关重要，糖原合酶激酶3β（GSK-3β）在足细胞中的作用较为复杂。深入了解GSK-3β对于理解糖尿病肾病的发病机制至关重要。为了分析GSK-3β在足细胞中的作用，应用成簇规律间隔短回文重复序列（CRISPR）-CRISPR相关蛋白（Cas）9构建的GSK-3β敲低慢病毒来建立稳定细胞系。利用质谱法寻找差异表达蛋白。结果发现，与对照细胞相比，GSK-3β敲低细胞中有34种蛋白表达水平升高，115种蛋白表达水平降低，并且鉴定出581个磷酸化水平升高的磷酸位点和288个磷酸化水平降低的磷酸位点。我们基于公共数据库对这些蛋白和磷酸化蛋白进行了功能富集分析。富集分析显示，GSK-3β参与剪接体、Hippo信号通路、肌动蛋白结合、结构分子活性等途径。然后，我们对磷酸化位点进行基序分析，基于1068个磷酸丝氨酸（pS）位点确定了89个保守基序，基于104个磷酸苏氨酸（pT）位点确定了15个保守基序。此外，使用STRING数据库进行蛋白质-蛋白质相互作用网络分析。利用Cytoscape的附加组件分子复合物检测（MCODE）来分析关键和核心蛋白组。在定量差异蛋白分析中，获得了4个MCODE，在差异表达蛋白的磷酸化蛋白质组分析中获得了22个MCODE。最后，我们分析了GSK-3β敲低后足细胞中的激酶调控网络，鉴定出299种蛋白激酶以及1574种蛋白上3460个显著变化的磷酸化修饰位点。这些结果对于进一步研究GSK-3β具有重要价值。
