Identification of SUMO Binding Enriched after Covalent Photo-Cross-Linking Proteins
Identification of SUMO Binding Enriched after Covalent Photo-Cross-Linking Proteins
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DOI：
10.1021/acschembio.0c00609
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发表时间：
2020-09-18
影响因子：
4
通讯作者：
Mootz, Henning D.
中科院分区：
文献类型：
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作者：
Bruninghoff, Kira;Aust, Annika;Mootz, Henning D.
小泛素样修饰物（SUMO）的翻译后修饰影响人类蛋白质组中的数千种蛋白质，并涉及许多细胞过程。SUMO结合的主要结果是通过SUMO结合蛋白识别表面修饰剂来重新连接蛋白质-蛋白质相互作用。SUMO相互作用基序（SIM）介导与SUMO上的凹槽结合;然而，这种相互作用的低亲和力和SIM序列的保守性差使SIM蛋白的分离和鉴定复杂化。为了解决这些挑战，我们设计并生化表征了单体和多聚体SUMO-2探针，其具有位于SIM结合沟旁边的遗传编码的光交联剂。在光诱导的共价捕获之后，即使是弱的SUMO结合剂在富集过程中也不会被洗掉，并且可以应用非常严格的洗涤条件来去除非特异性结合蛋白。共329个蛋白质从核HeLa细胞提取物中分离，并使用质谱鉴定。我们发现我们的探针的分子设计被许多已建立的SUMO相互作用蛋白的存在和高百分比（>90%）的含有潜在SIM序列的命中所证实，如生物信息学分析所预测的。值得注意的是，329种蛋白质中有266种以前没有报道过使用传统的非共价富集方法作为SUMO结合剂。我们证实了SUMO与纯化蛋白的结合，并绘制了所选病例的共价交联位置。我们假设MRE 11中有一个新的SIM，参与DNA修复。确定的SUMO结合候选物将有助于揭示复杂的SUMO介导的蛋白质网络。
Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of proteinprotein interactions through recognition of the modifiers surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.