Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China.
Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China; CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
Aquat Toxicol. 2018 Nov;204:180-189. doi: 10.1016/j.aquatox.2018.09.012. Epub 2018 Sep 20.
Sulfide-quinone oxidoreductase (SQR) is a key enzyme of sulfide metabolism in metazoans, and responsible for oxidizing sulfide into thiosulfate and transmitting the generated electrons to the ubiquinone. It has been revealed that the sqr mRNA level increases significantly in echiuran worm Urechis unicinctus exposed to sulfide, and HSF1, NF1 and Sp1 have been verified to participate in its transcriptional regulation. In this study, we obtained 23 potential transcription factors interacting possibly with the proximal region (-391 to +50) of sqr promoter, and focused on the RWD domain-containing 1 (Rwdd1), a protein with the maximum number of clones in yeast one-hybrid (Y1H) screening, to investigate its transcriptional regulation to U. unincitus sqr. The ChIP and EMSA assays identified that the Rwdd1 can bind directly to the promoter (+18/+36) of U. unicinctus sqr. The point mutation and transient transfection experiments discovered that TACG was the key sequence of the DNA element bound by the Rwdd1. Furthermore, the U. unicinctus Rwdd1 (UuRwdd1) was identified to be a transcription repressor inhibiting the sqr promoter activity, and the SUMOylation of UuRwdd1 at the lysine of 90 enhanced its inhibitory effect on sqr transcription further. Western blotting found Rwdd1 responded to sulfide in hindguts from U. unincitus, and the protein content showed a remarkable drop in hindgut nuclei in the early sulfide exposure, and then increased significantly both in the total protein and the nuclear protein extract. We suggested that the Rwdd1 is a novel transcription factor, and these data improve our understanding of the sqr transcriptional regulation and the mitochondrial sulfide metabolism.
硫化物-醌氧化还原酶（SQR）是后生动物硫化物代谢的关键酶，负责将硫化物氧化为硫代硫酸盐，并将产生的电子传递给泛醌。研究表明，在暴露于硫化物的文昌鱼 Urechis unicinctus 中，sqr mRNA 水平显著增加，并且已经验证了 HSF1、NF1 和 Sp1 参与其转录调控。在这项研究中，我们获得了 23 个可能与 sqr 启动子近端区域（-391 至+50）相互作用的潜在转录因子，并专注于 RWD 结构域包含 1（Rwdd1），这是酵母单杂交（Y1H）筛选中克隆数量最多的蛋白质，以研究其对 U. unincitus sqr 的转录调控。ChIP 和 EMSA 检测鉴定出 Rwdd1 可以直接结合 U. unicinctus sqr 启动子（+18/+36）。点突变和瞬时转染实验发现 TACG 是 Rwdd1 结合的 DNA 元件的关键序列。此外，鉴定出文昌鱼 Rwdd1（UuRwdd1）是一种转录抑制子，抑制 sqr 启动子活性，并且 UuRwdd1 在赖氨酸 90 处的 SUMO 化进一步增强了其对 sqr 转录的抑制作用。Western blot 发现 Rwdd1 对文昌鱼后肠中的硫化物有反应，并且在早期暴露于硫化物时，后肠细胞核中的蛋白质含量明显下降，随后在总蛋白和核蛋白提取物中均显著增加。我们认为 Rwdd1 是一种新型转录因子，这些数据提高了我们对 sqr 转录调控和线粒体硫化物代谢的理解。
