Aileni Vinay Kumar, Davydova Erna, Moen Anders, Falnes Pål Ø
Department of Biosciences, University of Oslo , P.O. Box 1066, Blindern, 0316 Oslo, Norway.
ACS Omega. 2017 Feb 28;2(2):462-469. doi: 10.1021/acsomega.6b00486. Epub 2017 Feb 10.
A number of lysine-specific methyltransferases (KMTs) are responsible for the post-translational modification of cellular proteins on lysine residues. Most KMTs typically recognize specific motifs in unstructured, short peptide sequences. However, we have recently discovered a novel KMT that appeared to have a more relaxed sequence specificity, namely, valosin-containing protein (VCP)-KMT, which trimethylates Lys-315 in the molecular chaperone VCP. On the basis of this, here, we explored the possibility of using the VCP-KMT/VCP system to obtain specific lysine methylation of desired sequences grafted onto a VCP-derived scaffold. We generated VCP-derived proteins in which three amino acid residues on each side of Lys-315 had been replaced by various sequences representing lysine methylation sites in histone H3. We found that all of these chimeric proteins were subject to efficient VCP-KMT-mediated methylation in vitro, and methylation was also observed in mammalian cells. Thus, we here describe a versatile system for introducing lysine methylation into a desired peptide sequence, and the approach should be readily expandable for generating combinatorial libraries of methylated sequences.
许多赖氨酸特异性甲基转移酶（KMTs）负责细胞蛋白赖氨酸残基的翻译后修饰。大多数KMTs通常识别无结构的短肽序列中的特定基序。然而，我们最近发现了一种新型的KMT，即含缬酪肽蛋白（VCP）-KMT，其序列特异性似乎更为宽松，它可使分子伴侣VCP中的赖氨酸-315发生三甲基化。基于此，我们在此探索了利用VCP-KMT/VCP系统对嫁接到VCP衍生支架上的所需序列进行特异性赖氨酸甲基化的可能性。我们生成了VCP衍生蛋白，其中赖氨酸-315两侧的三个氨基酸残基已被代表组蛋白H3中赖氨酸甲基化位点的各种序列所取代。我们发现所有这些嵌合蛋白在体外都能被VCP-KMT高效介导甲基化，并且在哺乳动物细胞中也观察到了甲基化现象。因此，我们在此描述了一种将赖氨酸甲基化引入所需肽序列的通用系统，并且该方法应该很容易扩展以生成甲基化序列的组合文库。
