School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Glasgow, G61 1QH, UK.
School of Molecular Biosciences, University of Glasgow, Glasgow, G12 8QQ, UK.
BMC Biol. 2022 Oct 8;20(1):228. doi: 10.1186/s12915-022-01415-y.
One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids.
We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, β-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels.
This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.
一碳代谢包括叶酸和蛋氨酸循环，涉及甲基的转移，然后被用作包括氧化还原防御在内的多种生理过程的一部分。在蛋氨酸循环中，维生素 B12 依赖性酶蛋氨酸合成酶将同型半胱氨酸转化为蛋氨酸。然后，酶 S-腺苷甲硫氨酸（SAM）合成酶利用蛋氨酸产生反应性甲基载体 SAM。SAM 结合甲基转移酶然后利用 SAM 作为辅因子将蛋白质、小分子、脂质和核酸甲基化。
我们描述了一种新的 SAM 甲基转移酶 RIPS-1，它是在秀丽隐杆线虫中进行正向遗传筛选以寻找对硫醇还原剂二硫苏糖醇（DTT）致死浓度的抗性时发现的单个基因。除了 RIPS-1 突变外，我们还表明，在野生型蠕虫中，通过调节维生素 B12 水平可以克服 DTT 毒性，方法是使用提供更高水平维生素 B12 的生长培养基和/或细菌食物，或通过补充维生素 B12。我们表明，活性蛋氨酸合成酶是野生型中维生素 B12 介导的 DTT 抗性所必需的，但不是由 RIPS-1 突变引起的抗性所必需的，并且蛋氨酸补充部分抑制了对 DTT 的敏感性。靶向 RNAi 修饰剂筛选确定了线粒体酶甲基丙二酰辅酶 A 差向异构酶是 RIPS-1 突变体中 DTT 抗性的强烈遗传增强剂。我们表明 RIPS-1 在线虫的肠道和皮下组织中表达，并且用 DTT、β-巯基乙醇或硫化氢处理诱导 RIPS-1 表达。我们证明 RIPS-1 表达受缺氧诱导因子途径控制，并且 RIPS-1 的同源物存在于一小部分真核生物和细菌中，其中许多可以适应环境氧水平的波动。
这项工作强调了膳食维生素 B12 在秀丽隐杆线虫正常代谢过程中的核心重要性，定义了这种维生素在对抗还原性应激中的新作用，并确定 RIPS-1 为蛋氨酸循环中的一种新的甲基转移酶。
