Profiling and Validation of Live-Cell Protein Methylation with Engineered Enzymes and Methionine Analogues.
Profiling and Validation of Live-Cell Protein Methylation with Engineered Enzymes and Methionine Analogues.
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DOI：
10.1002/cpz1.213
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发表时间：
2021-08
期刊：
影响因子：
--
通讯作者：
Luo M
中科院分区：
文献类型：
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作者：
Weiss N;Seneviranthe C;Jiang M;Wang K;Luo M
蛋白甲基转移酶（pmt）通过底物甲基化调节正常和疾病过程的许多方面，s -腺苷- l-蛋氨酸（SAM）是辅助因子。揭示细胞蛋白赖氨酸和精氨酸甲基化一直具有挑战性，因为这些修饰几乎不会改变靶蛋白的物理性质，而且通常是上下文依赖的、短暂的和亚化学计量的。为了揭示天然环境下与特定PMT活性相关的真正甲基化事件，我们开发了活细胞蛋白质甲基化生物正交分析（lcBPPM）技术，其中特定PMT的底物被活细胞内的工程PMT标记，原位合成的SAM类似物作为辅因子。该技术的双正交性是由于这些SAM模拟辅因子只能由工程pmt处理，而不是由天然pmt处理，从而用不同的化学基团修饰底物。在这里，我们描述了最新的lcBPPM协议及其在揭示蛋白质组范围甲基化和验证特定甲基化事件方面的应用。
Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to reveal cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context-dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analgoue cofactors can only be processed by the engineered PMTs —but not native PMTs — to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events.