摘要/Abstract

摘要： 目的 ·探讨真核细胞中组蛋白 H3K14M突变对嗜肺军团菌分泌的甲基转移酶 RomA活性的影响及其内在机制。方法 ·构建组蛋白 H3野生型（ H3WT）以及组蛋白 H3上 14位赖氨酸突变为甲硫氨酸、异亮氨酸、精氨酸（ H3K14M、H3K14I、H3K14R）的真核表达质粒，慢病毒包装并感染稳定表达 RomA的 293T、THP-1等细胞。用 Western blotting检测并比较带有不同 H3K14突变的细胞中内源 H3K14位点表观修饰的改变。并利用免疫共沉淀技术检测 H3K14M与 RomA蛋白的相互作用。结果 ·嗜肺军团菌的分泌效应物 RomA通过靶向宿主细胞核，增加其 H3K14甲基化水平同时降低 H3K14乙酰化水平，从而抑制宿主基因的表达，促进嗜肺军团菌有效的细胞内复制；但组蛋白 H3K14M突变可以通过增强与 RomA蛋白的相互作用以抑制其甲基转移酶活性，阻断 RomA对 H3K14位点的甲基化。结论 ·组蛋白 H3K14M突变显著抑制嗜肺军团菌来源甲基转移酶 RomA活性。
关键词: 嗜肺军团菌, RomA, SET结构域, H3K14M, 翻译后修饰
Abstract:
Objective · To investigate the effect of histone H3K14M mutation on the activity of methyltransferase RomA, a secreted effector of Legionella pneumophila in eukaryotic cells and the underlying mechanisms. Methods · Wide-type histone H3 (H3WT) and mutant histone H3 (the lysine residue 14 was replacedmethionine, isoleucine or arginine residue, and named as H3K14M, H3K14I, and H3K14R, respectively) recombinant plasmids were constructed. Packaged lentiviruses with these plasmids were used to infect eukaryotic cells 293T and THP-1 with or without over- of RomA. The H3K14 methylation and acetylation were analyzedWestern blotting. The interaction of RomA with H3WT and H3K14 mutants was detectedco-immunoprecipitation. Results · A secreted effector of Legionella pneumophila named RomA targeted the host cell nucleus to upregulate the H3K14 methylation level and downregulate the H3K14 acetylation level for inhibiting the gene in host cells and promoting Legionella pneumophila’s efficient intracellular replication. But histone H3K14M mutation could promote the interaction between H3K14M and RomA and thus inhibited the methyltransferase activity of RomA. Conclusion · Histone H3K14M mutation significantly inhibits the activity of Legionella pneumophila methyltransferase RomA.
Key words: Legionella pneumophila, RomA, SET-domain, H3K14M, posttranslational modification


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