Yoshida Yutaka, Nishiyama Akihito, Suameitria Dewi Desak Nyoman Surya, Yamazaki Tomoya, Yokoyama Akira, Kobayashi Daiki, Kondo Hitoshi, Ozeki Yuriko, Matsumoto Sohkichi
Department of Bacteriology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Chuo-Ku, Niigata, 951-8510, Japan.
Department of Bacteriology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Chuo-Ku, Niigata, 951-8510, Japan.
Biochem Biophys Res Commun. 2023 Nov 12;681:111-119. doi: 10.1016/j.bbrc.2023.09.028. Epub 2023 Sep 20.
The basic, intrinsically disordered regions of eukaryotic histones and their bacterial counterparts are presumed to act as signaling hubs to regulate the compaction of chromosomes or nucleoids and various DNA processes such as gene expression, recombination, and DNA replication. Posttranslational modifications (PTMs) on these regions are pivotal in regulating chromosomal or nucleoid compaction and DNA processes. However, the low sequence complexity and the presence of short lysine-rich repeats in the regions have hindered the accurate determination of types and locations of PTMs using conventional proteomic procedures. We described a limited proteolysis protocol using trypsin to analyze PTMs on mycobacterial DNA-binding protein 1 (MDP1), a nucleoid-associated protein in mycobacterial species that possesses an extended, lysine-rich, intrinsically disordered region in its C-terminal domain. This limited proteolysis approach successfully revealed significant methylation on many lysine residues in the C-terminal domain of MDP1 purified from Mycobacterium tuberculosis, which was lacking in the corresponding region of recombinant MDP1 expressed in Escherichia coli.
真核生物组蛋白及其细菌对应物的基本内在无序区域被认为可作为信号枢纽，以调节染色体或类核的压缩以及各种DNA相关过程，如基因表达、重组和DNA复制。这些区域的翻译后修饰（PTM）对于调节染色体或类核压缩以及DNA相关过程至关重要。然而，这些区域的低序列复杂性以及富含赖氨酸的短重复序列的存在，阻碍了使用传统蛋白质组学方法准确确定PTM的类型和位置。我们描述了一种使用胰蛋白酶的有限蛋白酶解方案，用于分析分枝杆菌DNA结合蛋白1（MDP1）上的PTM，MDP1是分枝杆菌属中的一种类核相关蛋白，其C端结构域具有一个延伸的、富含赖氨酸的内在无序区域。这种有限蛋白酶解方法成功揭示了从结核分枝杆菌纯化的MDP1的C端结构域中许多赖氨酸残基上的显著甲基化，而在大肠杆菌中表达的重组MDP1的相应区域中则没有这种甲基化。
