Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA; Stark Neuroscience Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA; Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana, USA.
J Biol Chem. 2023 May;299(5):104651. doi: 10.1016/j.jbc.2023.104651. Epub 2023 Mar 25.
Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.
赖氨酸甲基化是一种动态的翻译后修饰标记，可调节组蛋白和非组蛋白蛋白的功能。许多介导赖氨酸甲基化的酶，称为赖氨酸甲基转移酶（KMTs），最初被鉴定为修饰组蛋白，但也被发现可甲基化非组蛋白。在这项工作中，我们研究了 KMT PRDM9 的底物选择性，以鉴定潜在的组蛋白和非组蛋白底物。尽管 PRDM9 通常在生殖细胞中表达，但在许多癌症类型中其表达水平显著上调。PRDM9 的甲基转移酶活性对于减数分裂重组过程中的双链断裂形成至关重要。据报道，PRDM9 可在组蛋白 H3 的赖氨酸残基 4 和 36 处甲基化；然而，PRDM9 KMT 活性以前并未在非组蛋白蛋白上进行评估。我们使用针对赖氨酸的肽文库筛选 PRDM9 的潜在底物，确定 PRDM9 优先甲基化在任何组蛋白蛋白中都找不到的肽序列。我们通过在关键位置进行取代的肽的体外 KMT 反应确认了 PRDM9 的选择性。多点 λ动力学计算分析为观察到的 PRDM9 选择性提供了结构依据。然后，使用底物选择性图谱来鉴定推定的非组蛋白底物，并用肽斑点阵列进行测试，并通过体外 KMT 测定法在重组蛋白上对蛋白质水平的一小部分进行进一步验证。最后，发现非组蛋白底物之一 CTNNBL1 可被 PRDM9 在细胞中甲基化。
