在DNMT2/TRDMT1中通过分子间二硫化物键进行受限制的tRNA甲基化
Huari Li1, Daiyun Zhu1, Yapeng Yang1
1College of Veterinary Medicine, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan 430070, Hubei, China.
International journal of biological macromolecules
|August 14, 2023
概括
DNMT2/TRDMT1酶活性受到二硫化键的限制,影响tRNA甲基化. 了解这种机制对于与氧化还原压力相关的疾病至关重要.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
背景情况:
- DNMT2/TRDMT1在C38处甲基化tRNA,这对生存和恒温至关重要.
- 与TrmD和Trm5.5相比,DNMT2/TRDMT1在体外表现出较弱的tRNA甲基化活性.
研究的目的:
- 研究限制DNMT2/TRDMT1的tRNA甲基化活性的机制.
- 阐明二硫化物键在DNMT2/TRDMT1功能和调节中的作用.
主要方法:
- 在优化的缓冲条件下 (缓冲C,37°C) 用Dithiothreitol (DTT) 进行酶活性测定.
- 使用减少剂分析蛋白质构成和寡合体形成.
- 液体染色学-双重质谱学 (LC-MS/MS) 用于识别二硫化物键.
- 在不同压力条件下对HEK293T细胞的表达分析 (GSSG,GSH).
主要成果:
- 优化条件和DTT对于DNMT2/TRDMT1激活至关重要.
- 确定了主要的分子间二硫化键:在重组蛋白中C292-C292,C292-C287;在HEK293T细胞中C79-C24,C292-C292,C222-C24,形成二聚体.
- GSSG压力最初增强了tRNA甲基化,但后来变得不利于酶核积累.
- 压力GSH降低了DNMT2/TRDMT1的表达,并促进了tRNA甲基化,可能是通过破坏二硫化物键.
结论:
- 二硫化键限制了DNMT2/TRDMT1tRNA的甲基化活动.
- 这些发现提供了对tRNA甲基化的氧化还原应激调节的见解.
- 对理解和治疗与氧化还原压力相关的疾病的影响.
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