Abstract

SUMOylation of proteins regulates cell behaviors and is reversibly removed by small ubiquitin-like modifier (SUMO)-specific proteases (SENPs). The SENP family member SENP3 is involved in SUMO2/3 deconjugation and has been reported to sense cell stress and accumulate in several human cancer cells and macrophages. We previously reported that Senp3-knockout heterozygous mice showed smaller liver, but the pertinent mechanisms of SENP3 and SUMOylated substrates remain unclear. Thus, in this study, we investigated the interacting proteins with SENP3 and the alteration in hepatocytes treated with the xenobiotic diethylnitrosamine (DEN), which is specifically transformed in the liver and induces DNA double-strand breaks. Our data revealed that a certain amount of SENP3 was present in normal, untreated hepatocytes; however, DEN treatment promoted rapid SENP3 accumulation. SENP3 was mainly localized in the nuclei, and its level was significantly increased in the cytoplasm after 2 h of DEN treatment. The application of the recent proximity-dependent biotinylation (BioID) method led to the identification of 310 SENP3-interacting proteins that were involved in not only gene transcription but also RNA splicing, protein folding, and metabolism. Furthermore, after DEN exposure for a short duration, ribosomal proteins as well as proteins associated with mitochondrial ATP synthesis, membrane transport, and bile acid synthesis, rather than DNA repair proteins, were identified. This study provides insights into the diverse regulatory roles of SENP3, and the BioID method seems to be efficient for identifying physiologically relevant insoluble proteins.

中文翻译：

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BioID 法评估二乙基亚硝胺处理的肝细胞中 SENP3 相互作用蛋白

摘要

蛋白质的 SUMO 化可调节细胞行为，并被小泛素样修饰剂 (SUMO) 特异性蛋白酶 (SENP) 可逆地去除。SENP 家族成员 SENP3 参与 SUMO2/3 去结合，据报道可感知细胞压力并在几种人类癌细胞和巨噬细胞中积累。我们之前报道过Senp3敲除杂合子小鼠肝脏较小，但 SENP3 和 SUMO 化底物的相关机制仍不清楚。因此，在这项研究中，我们研究了与 SENP3 相互作用的蛋白质以及用异生物质二乙基亚硝胺 (DEN) 处理的肝细胞的改变，DEN 在肝脏中特异性转化并诱导 DNA 双链断裂。我们的数据显示，在正常、未经处理的肝细胞中存在一定量的 SENP3。然而，DEN 处理促进了 SENP3 的快速积累。SENP3主要定位于细胞核中，在DEN处理2小时后其在细胞质中的水平显着升高。最近的邻近依赖性生物素化 (BioID) 方法的应用导致鉴定了 310 种 SENP3 相互作用蛋白，这些蛋白不仅参与基因转录，还参与 RNA 剪接、蛋白质折叠和代谢。此外，在短时间的 DEN 暴露后，核糖体蛋白以及与线粒体 ATP 合成、膜转运和胆汁酸合成相关的蛋白质，而不是 DNA 修复蛋白被鉴定出来。这项研究提供了对 SENP3 不同调节作用的见解，并且 BioID 方法似乎可以有效地识别生理相关的不溶性蛋白质。和胆汁酸合成，而不是 DNA 修复蛋白，被确定。这项研究提供了对 SENP3 不同调节作用的见解，并且 BioID 方法似乎可以有效地识别生理相关的不溶性蛋白质。和胆汁酸合成，而不是 DNA 修复蛋白，被确定。这项研究提供了对 SENP3 不同调节作用的见解，并且 BioID 方法似乎可以有效地识别生理相关的不溶性蛋白质。