Molecular Pharmacology and Chemistry Program and ‡Tri-Institutional Training Program in Chemical Biology, Memorial Sloan-Kettering Cancer Center , New York, New York 10065, United States.
ACS Chem Biol. 2014 Feb 21;9(2):476-84. doi: 10.1021/cb4008259. Epub 2013 Dec 9.
Protein arginine N-methyltransferase 3 (PRMT3) belongs to the family of type I PRMTs and harbors the activity to use S-adenosyl-l-methionine (SAM) as a methyl-donor cofactor for protein arginine labeling. However, PRMT3's functions remain elusive with the lacked knowledge of its target scope in cellular settings. Inspired by the emerging Bioorthogonal Profiling of Protein Methylation (BPPM) using engineered methyltransferases and SAM analogues for target identification, the current work documents the endeavor to systematically explore the SAM-binding pocket of PRMT3 and identify suitable PRMT3 variants for BPPM. The M233G single point mutation transforms PRMT3 into a promiscuous alkyltransferase using sp(2)-β-sulfonium-containing SAM analogues as cofactor surrogates. Here the conserved methionine was defined as a hot spot that can be engineered alone or in combination with nearby residues to render cofactor promiscuity of multiple type I PRMTs. With this promiscuous variant and the matched 4-propargyloxy-but-2-enyl (Pob)-SAM analogue as the BPPM reagents, more than 80 novel proteins were readily uncovered as potential targets of PRMT3 in the cellular context. Subsequent target validation and functional analysis correlated the PRMT3 methylation to several biological processes such as cytoskeleton dynamics, whose roles might be compensated by other PRMTs. These BPPM-revealed substrates are primarily localized but not restricted in cytoplasm, the preferred site of PRMT3. The broad localization pattern may implicate the diverse roles of PRMT3 in the cellular setting. The revelation of PRMT3 targets and the transformative character of BPPM for other PRMTs present unprecedented pathways toward elucidating physiological and pathological roles of diverse PRMTs.
精氨酸 N-甲基转移酶 3（PRMT3）属于 I 型 PRMT 家族，具有使用 S-腺苷甲硫氨酸（SAM）作为蛋白质精氨酸标记的甲基供体辅助因子的活性。然而，由于缺乏细胞环境中 PRMT3 靶标范围的知识，其功能仍然难以捉摸。受使用工程甲基转移酶和 SAM 类似物进行蛋白质甲基化的新兴生物正交分析（BPPM）的启发，本研究旨在系统探索 PRMT3 的 SAM 结合口袋，并鉴定合适的 PRMT3 变体用于 BPPM。M233G 单点突变将 PRMT3 转化为一种利用 sp(2)-β-硫代磺酸盐 SAM 类似物作为辅助因子替代物的广谱烷基转移酶。这里将保守的蛋氨酸定义为一个热点，可以单独或与附近的残基组合进行工程改造，从而使多种 I 型 PRMT 的辅助因子具有广谱性。利用这种广谱变体和匹配的 4-炔丙氧基丁-2-烯基（Pob）-SAM 类似物作为 BPPM 试剂，在细胞环境中，很容易发现 80 多种新的蛋白质可能是 PRMT3 的潜在靶标。随后的靶标验证和功能分析将 PRMT3 甲基化与几种生物学过程相关联，如细胞骨架动力学，其作用可能被其他 PRMT 补偿。这些 BPPM 揭示的底物主要定位于细胞质中，但不限于细胞质，这是 PRMT3 的首选位置。广泛的定位模式可能暗示 PRMT3 在细胞环境中的多种作用。PRMT3 靶标的揭示和 BPPM 对其他 PRMT 的变革性特征为阐明不同 PRMT 的生理和病理作用提供了前所未有的途径。
