Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
J Am Chem Soc. 2012 Apr 4;134(13):5909-15. doi: 10.1021/ja2118333. Epub 2012 Mar 26.
Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2's methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.
蛋白质甲基转移酶（PMTs）在多种生物过程中发挥着关键作用。由于 PMTs 通常通过形成多聚体蛋白复合物在体内发挥作用，因此在天然环境中解析它们的活性具有挑战性但又很重要。为了满足这一需求，我们设想了一种生物正交的蛋白质甲基化分析（BPPM）技术，其中 SAM 类似物辅因子可以被多个合理设计的 PMTs 利用，以标记相应天然 PMTs 的底物。在这里，S-腺苷甲硫氨酸的 4-叠氮丁-2-烯基衍生物（Ab-SAM）被报道为一种合适的 BPPM 辅因子。所得的辅因子-酶对被用于在复杂的细胞混合物中特异性标记密切相关的 PMTs（如 EuHMT1 和 EuHMT2）的底物。BPPM 方法结合质谱分析，能够鉴定 EuHMT1/2 的非组蛋白靶标。EuHMT1/2 的甲基组学比较表明，虽然这两种人类 PMTs 在一级序列上相似，但它们可以作用于不同的非组蛋白靶标。鉴于 PMTs 的保守活性位点，Ab-SAM 及其在 BPPM 中的应用有望被转移到其他 PMTs 中用于靶标鉴定。
