Effects of Salicylate Derivatives on Localization of p.H723R Pendrin Mutant.
Effects of Salicylate Derivatives on Localization of p.H723R Pendrin Mutant.
复制标题
水杨酸酯衍生物对 p.H723R Pendrin 突变体定位的影响。
DOI：
10.1016/j.anl.2022.03.009
复制
发表时间：
2022
影响因子：
1.7
通讯作者：
Wada H.
中科院分区：
文献类型：
--
作者：
Murakoshi M;Koyama S;Kobayashi T;Usami S;Kamiya K;Ikeda K;Haga Y;Tsumoto K;Nakamura H;Hirasawa N;Ishihara K;Wada H.
目的Pendrin是一种由SLC26A4基因编码的跨膜蛋白，其功能是维持内耳内淋巴中的离子浓度，最可能的作用是作为氯/碳酸氢盐转运体。SLC26A4基因的变异是感觉神经性听力损失的原因。虽然膜蛋白定位于质膜，但我们先前发现SLC26A4的8个错义等位基因产物保留在细胞内区，失去了阴离子交换功能。我们还发现，10 mM水杨酸盐能诱导细胞内区8个等位基因产物中的4个移位到质膜上，并恢复它们的阴离子交换活性。然而，由于10 mM水杨酸盐具有细胞毒性，需要使用细胞毒性较小的化合物。因此，本研究以水杨酸衍生物为化合物，研究其对SLC26A4的P.H723R等位基因产物的影响。通过细胞增殖、细胞活力和细胞毒性实验，观察4种水杨酸衍生物(2-羟基苯甲醇、2，3-二羟基苯甲酸、2‘-羟基苯乙酮和水杨酸甲酯)处理培养细胞后的反应和细胞健康状况。结果加入10 mM水杨酸盐后，细胞毒性增加，细胞存活率下降，细胞增殖能力明显下降。相反，应用1 mM水杨酸衍生物并没有显示出任何显著的细胞毒性增加和细胞活力下降，相应地，细胞浓度随着培养时间的增加而对数增加。免疫荧光实验表明，P.H723R保留在内质网中。在应用的水杨酸衍生物中，2-羟基苯甲醇在作用3h后可诱导P.H723R从内质网向质膜移位。结论2-羟基苯甲醇以1 mM的浓度在作用3h后恢复了SLC26A4的p.H723R等位基因产物从内质网到质膜的定位，其细胞毒性低于10 mM水杨酸盐。
ObjectivePendrin is a transmembrane protein encoded by theSLC26A4gene that functions in maintaining ion concentrations in the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Variants in theSLC26A4gene are responsible for sensorineural hearing loss. Although pendrin localizes to the plasma membrane, we previously identified that 8 missense allele products ofSLC26A4were retained in the intracellular region and lost their anion exchange function. We also found that 10 mM salicylate induced the translocation of 4 out of 8 allele products from the intracellular region to the plasma membrane and restored their anion exchanger activity. However, since 10 mM salicylate exhibits cytotoxicity, the use of chemical compounds with less cell toxicity is needed. In the present study, therefore, salicylate derivatives were used as the chemical compounds and their effects on the p.H723R allele products ofSLC26A4were investigated.MethodsHEK293 cells were transfected with the cDNA of p.H723R. Cell proliferation, viability and toxicity assays were performed to investigate the response and health of cells in culture after treatment with four types of salicylate derivatives, i.e., 2-hydroxybenzyl alcohol, 2,3-dihydroxybenzoic acid, 2’-hydroxyacetophenone and methyl salicylate. The effects of these salicylate derivatives on the localization of the p.H723R were investigated by immunofluorescence microscopy.ResultsThe application of 10 mM salicylate showed an increase in cell toxicity and decrease in cell viability, leading to a significant decrease in cell proliferation. In contrast, the application of 1 mM salicylate derivatives did not show any significant increase in cell toxicity and decrease in cell viability, corresponding to a logarithmic increase in cell concentration with an increase in culture time. Immunofluorescence experiments showed that the p.H723R retained in the endoplasmic reticulum (ER). Among the salicylate derivatives applied, 2-hydroxybenzyl alcohol induced the translocation of p.H723R from the ER to the plasma membrane 3 h after its application.ConclusionThe results obtained showed that 2-hydroxybenzyl alcohol restored the localization of the p.H723R allele products ofSLC26A4from the ER to the plasma membrane at a concentration of 1 mM by 3 h after its administration with less cytotoxicity than 10 mM salicylate.