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<p>Lysine acylation is a ubiquitous post-translational modification (PTM) that plays pivotal roles in various cellular processes, such as transcription, metabolism, protein localization and folding. Thousands of lysine acylation sites have been identified based on advances in antibody enrichment strategies, highly sensitive analysis by mass spectrometry (MS), and bioinformatics. However, only 27 lysine methacrylation (Kmea) sites have been identified exclusively in histone proteins. It is hard to separate, purify and differentiate the Kmea modification from its structural isomer lysine crotonylation (Kcr) using general biochemical approaches. Here, we identify Kmea sites on a non-histone protein, Cyclophillin A (CypA). To investigate the functions of Kmea in CypA, we develop a general genetic code expansion approach to incorporate a non-canonical amino acid (ncAA) <i>ε</i>-<i>N</i>-Methacryllysine (MeaK) into target proteins and identify interacting proteins of methacrylated CypA using affinity-purification MS. We find that Kmea at CypA site 125 regulates cellular redox homeostasis, and HDAC1 is the regulator of Kmea on CypA. Moreover, we discover that genetically encode Kmea can be further methylated to <Emphasis Type="BoldItalic">ε</Emphasis><b>-</b><i>N</i>-methyl-<Emphasis Type="BoldItalic">ε</Emphasis><b>-</b><i>N</i>-methacrylation (Kmemea) in live cells.</p>

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Genetically encoding ε-N-methacryllysine into proteins in live cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月17日

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  • Tian-Yi Zhu,
  • Shi-Yi Chen,
  • Mengdi Zhang,
  • Heyu Li,
  • Ting Wu,
  • Emmanuel Ajiboye,
  • Jia Wen Wang,
  • Bi-Kun Jin,
  • Dan-Dan Liu,
  • Xintong Zhou,
  • He Huang,
  • Xiaobo Wan,
  • Ke Sun,
  • Peilong Lu,
  • Yaxin Fu,
  • Ying Yuan,
  • Hai Song,
  • Anna A. Sablina,
  • Chao Tong,
  • Long Zhang,
  • Ming Wu,
  • Haifan Wu,
  • Bing Yang

摘要

Lysine acylation is a ubiquitous post-translational modification (PTM) that plays pivotal roles in various cellular processes, such as transcription, metabolism, protein localization and folding. Thousands of lysine acylation sites have been identified based on advances in antibody enrichment strategies, highly sensitive analysis by mass spectrometry (MS), and bioinformatics. However, only 27 lysine methacrylation (Kmea) sites have been identified exclusively in histone proteins. It is hard to separate, purify and differentiate the Kmea modification from its structural isomer lysine crotonylation (Kcr) using general biochemical approaches. Here, we identify Kmea sites on a non-histone protein, Cyclophillin A (CypA). To investigate the functions of Kmea in CypA, we develop a general genetic code expansion approach to incorporate a non-canonical amino acid (ncAA) ε-N-Methacryllysine (MeaK) into target proteins and identify interacting proteins of methacrylated CypA using affinity-purification MS. We find that Kmea at CypA site 125 regulates cellular redox homeostasis, and HDAC1 is the regulator of Kmea on CypA. Moreover, we discover that genetically encode Kmea can be further methylated to ε-N-methyl-ε-N-methacrylation (Kmemea) in live cells.