Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka-shi, Fukuoka 812-8582, Japan.
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka-shi, Fukuoka 812-8582, Japan; Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka-shi, Fukuoka 812-8582, Japan.
Mitochondrion. 2020 Jul;53:99-108. doi: 10.1016/j.mito.2020.05.003. Epub 2020 May 18.
Chemical acetylation is postulated to occur in mitochondria. Mitochondrial transcription factor A (TFAM or mtTFA), a mitochondrial transcription initiation factor as well as the major mitochondrial nucleoid protein coating the entire mitochondrial genome, is proposed to be acetylated in animals and cultured cells. This study investigated the properties of human TFAM, in conjunction with the mechanism and effects of TFAM acetylation in vitro. Using highly purified recombinant human TFAM and 3 kb circular DNA as a downsized mtDNA model, we studied how the global TFAM-DNA interaction is affected/regulated by the quantitative TFAM-DNA relationship and TFAM acetylation. Results showed that the TFAM-DNA ratio strictly affects the TFAM property to unwind circular DNA in the presence of topoisomerase I. Mass spectrometry analysis showed that in vitro chemical acetylation of TFAM with acetyl-coenzyme A occurs preferentially on specific lysine residues, including those reported to be acetylated in exogenously expressed TFAM in cultured human cells, indicating that chemical acetylation plays a crucial role in TFAM acetylation in mitochondria. Intriguingly, the modification significantly decreased TFAM's DNA-unwinding ability, while its DNA-binding ability was largely unaffected. Altogether, we propose TFAM is chemically acetylated in vivo, which could change mitochondrial DNA topology, leading to copy number and gene expression modulation.
化学乙酰化被假定发生在线粒体中。线粒体转录因子 A（TFAM 或 mtTFA）既是线粒体转录起始因子，也是覆盖整个线粒体基因组的主要线粒体核仁蛋白，据推测在动物和培养细胞中会发生乙酰化。本研究调查了人 TFAM 的特性，同时还研究了 TFAM 体外乙酰化的机制和影响。本研究使用高度纯化的重组人 TFAM 和 3kb 环状 DNA 作为小型化 mtDNA 模型，研究了定量 TFAM-DNA 关系和 TFAM 乙酰化如何影响/调节全局 TFAM-DNA 相互作用。结果表明，TFAM-DNA 比值严格影响 TFAM 在拓扑异构酶 I 存在的情况下解开环状 DNA 的特性。质谱分析表明，TFAM 与乙酰辅酶 A 的体外化学乙酰化优先发生在特定赖氨酸残基上，包括在培养的人类细胞中外源表达的 TFAM 中报道的那些乙酰化赖氨酸残基，表明化学乙酰化在 TFAM 在线粒体中的乙酰化中起着至关重要的作用。有趣的是，该修饰显著降低了 TFAM 的 DNA 解旋能力，而其 DNA 结合能力基本不受影响。总之，我们提出 TFAM 在体内发生化学乙酰化，这可能会改变线粒体 DNA 的拓扑结构，导致拷贝数和基因表达的调节。
