Abstract：
The ubiquitination function in diabetic nephropathy (DN) has attracted much attention, but there is a lack of information on its ubiquitylome profile. To examine the differences in protein content and ubiquitination in the kidney between db/db mice and db/m mice, we deployed liquid chromatography-mass spectrometry (LC-MS/MS) to conduct analysis. We determined 145 sites in 86 upregulated modified proteins and 66 sites in 49 downregulated modified proteins at the ubiquitinated level. Moreover, 347 sites among the 319 modified proteins were present only in the db/db mouse kidneys, while 213 sites among the 199 modified proteins were present only in the db/m mouse kidneys. The subcellular localization study indicated that the cytoplasm had the highest proportion of ubiquitinated proteins (31.87%), followed by the nucleus (30.24%) and the plasma membrane (20.33%). The enrichment analysis revealed that the ubiquitinated proteins are mostly linked to tight junctions, oxidative phosphorylation, and thermogenesis. Podocin, as a typical protein of slit diaphragm, whose loss is a crucial cause of proteinuria in DN. Consistent with the results of ubiquitination omics, the K261R mutant of podocin induced the weakest ubiquitination compared with the K301R and K370R mutants. As an E3 ligase, c-Cbl binds to podocin, and the regulation of c-Cbl can impact the ubiquitination of podocin. In conclusion, in DN, podocin ubiquitination contributes to podocyte injury, and K261R is the most significant site. c-Cbl participates in podocin ubiquitination and may be a direct target for preserving the integrity of the slit diaphragm structure, hence reducing proteinuria in DN.
摘要：
泛素化在糖尿病肾病(DN)中的作用备受关注,但是缺乏关于其泛素组的信息。为了检查db/db小鼠和db/m小鼠之间肾脏中蛋白质含量和泛素化的差异，我们使用液相色谱-质谱(LC-MS/MS)进行分析。我们在泛素化水平上确定了86个上调的修饰蛋白中的145个位点和49个下调的修饰蛋白中的66个位点。此外，319个修饰蛋白中的347个位点仅存在于db/db小鼠肾脏中，而199个修饰蛋白中的213个位点仅存在于db/m小鼠肾脏中。亚细胞定位研究表明，细胞质中泛素化蛋白的比例最高（31.87％），其次是细胞核(30.24%)和质膜(20.33%)。富集分析显示，泛素化的蛋白质主要与紧密连接相关，氧化磷酸化，和产热。Podocin,作为狭缝隔膜的典型蛋白质，其丢失是DN中蛋白尿的关键原因。与泛素化组学的结果一致，与K301R和K370R突变体相比，Podocin的K261R突变体诱导的泛素化作用最弱。作为E3连接酶，c-Cbl与足孔素结合,c-Cbl的调节可以影响podocin的泛素化。总之,在DN中，podocin泛素化有助于足细胞损伤，和K261R是最重要的网站。c-Cbl参与podocin泛素化，可能是保持狭缝隔膜结构完整性的直接靶标，从而减少DN的蛋白尿。
