Molecular Pharmacology and Chemistry Program and Tri-Institutional Training Program in Chemical Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.
Proc Natl Acad Sci U S A. 2013 Oct 15;110(42):16778-83. doi: 10.1073/pnas.1216365110. Epub 2013 Sep 30.
Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme-cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp(2) carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.
蛋白质甲基转移酶（PMT）介导的组蛋白和非组蛋白底物的翻译后修饰调节了靶蛋白在不同细胞环境中的稳定性、定位和相互作用伙伴。这些事件在正常的生物学过程中起着关键作用，并且在人类疾病中经常失调。在鉴定单个 PMT 的底物的过程中，生物正交的蛋白质甲基化谱分析（BPPM）已经证明了其价值。在这种方法中，特定的 PMT 被设计为处理 S-腺苷-L-蛋氨酸（SAM）类似物作为辅助因子的替代物，并使用不同的化学修饰标记其底物，以确定靶标。尽管 BPPM 的概念验证取得了进展，但很少有努力探索其普遍性。我们以两种与癌症相关的 PMT，EuHMT1（GLP1/KMT1D）和 EuHMT2（G9a/KMT1C）作为模型，定义了工程化 PMT 和匹配的 SAM 类似物的关键结构特征，这些特征可以为高效催化提供正交酶-辅助因子对。在这里，我们已经证明，硫翁-β-sp(2)碳和柔性、中等大小的硫翁-δ-取代基的存在对于作为 BPPM 试剂的 SAM 类似物是至关重要的。通过调整 EuHMT1/2 的保守 Y1211/Y1154 残基和附近的疏水腔，可以容纳大体积的辅助因子。使用 BPPM 对全蛋白质组范围的底物进行分析，鉴定了超过 500 个 EuHMT1/2 的靶点，并用天然 EuHMT1/2 和 SAM 对代表性靶点进行了验证。这一发现表明，EuHMT1/2 可能调节了许多以前未被认为受甲基化调节的细胞事件。因此，本工作为更广泛地应用 BPPM 技术来分析不同 PMT 的甲基组谱和阐明其下游功能铺平了道路。
