From the Department of Molecular Biophysics and Biochemistry , Yale University , New Haven , Connecticut 06520.
Department of Chemistry , Yale University , New Haven , Connecticut 06520.
Chem Res Toxicol. 2019 Nov 18;32(11):2305-2319. doi: 10.1021/acs.chemrestox.9b00315. Epub 2019 Oct 16.
Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.
生物体暴露在空气中、水中和土壤中的氟化物中。酵母和其他微生物利用氟化物通道作为一种防止细胞内氟化物积累和调节氟化物毒性的方法。因此，[生物体名称]中氟化物外排基因（FEX）的缺失导致氟化物敏感性增加了 1000 多倍。我们使用这种 FEX 敲除菌株来鉴定基因，这些基因在过表达时能够部分缓解氟化物暴露的毒性。过表达五个基因（SSU1、YHB1、IPP1、PHO87 和 PHO90）可将氟化物耐受性提高 2 到 10 倍。这些基因在野生型酵母中的过表达并没有提供更好的氟化物抗性，这表明该机制是特定于酵母中低氟化物毒性的。Ssu1p 和 Yhb1p 都在硝化应激反应中发挥作用，氟化物暴露会同时诱导金属流入并引发硝化应激反应。Ipp1p、Pho87p 和 Pho90p 增加细胞内正磷酸盐。这与观察结果一致，向生长培养基中添加高浓度磷酸盐也部分减轻了氟化物毒性。氟化物在应激诱导时抑制磷酸盐的摄取，导致营养饥饿和细胞器破坏，这得到了通过 RNA-Seq 监测的基因诱导的支持。这些观察结果表明，跨膜营养转运蛋白是氟化物引发应激时最敏感的蛋白质之一。
