Expression analysis and functional characterization of thioredoxin domain-containing protein 11
Expression analysis and functional characterization of thioredoxin domain-containing protein 11
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DOI：
10.1007/s11033-022-07932-x
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发表时间：
2022-09
影响因子：
2.8
通讯作者：
R. Murase;Ayumi Yamamoto;Y. Hirata;K. Oh‐hashi
中科院分区：
文献类型：
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作者：
R. Murase;Ayumi Yamamoto;Y. Hirata;K. Oh‐hashi
背景内质网（ER）是一个重要的细胞器，它调节许多蛋白质的折叠、修饰和运输，并感知细胞内外的某些刺激。 ER 相关降解 (ERAD)，包括 SEL1L，是维持体内平衡的重要机制。在本研究中，我们在野生型 (wt) 和 SEL1L 缺陷细胞中进行了比较蛋白质组分析。方法和结果通过 RT-PCR 和蛋白质印迹分析，我们发现在我们的 SEL1L 缺陷 HEK293 细胞中，硫氧还蛋白结构域蛋白 11 (TXNDC11) mRNA 和蛋白组成性高表达。 TXNDC11 基因在其转录起始位点周围拥有一个高度保守的未折叠蛋白反应元件 (UPRE)，ER 应激通过 HEK293 细胞中这个假定的 UPRE 增加了 TXNDC11 mRNA 和荧光素酶报告基因活性。经或未经毒胡萝卜素 (Tg) 处理的野生型和 SEL1L 缺陷细胞中 TXNDC11 蛋白的量与其在这些细胞中的 mRNA 平行，几乎与剪接的 XBP1 (sXBP1) mRNA 表达成正比。 TXNDC11 缺陷型 HEK293 细胞的建立和表征表明，三种不同的 ER 驻留应激传感器 ATF6α、CREB3 和 CREB3L2 的表达受 TXNDC11 调节。在wt细胞中CHX处理后三种蛋白的消失率明显不同，全长CREB3L2蛋白在CHX处理后15分钟内几乎完全降解。 TXNDC11缺陷在静息条件下增加了每种全长形式的表达，并通过CHX处理延迟了它们的消失。有趣的是，TXNDC11缺陷引起的全长CREB3/CREB3L2增加程度明显高于全长ATF6α。 TXNDC11 缺陷导致的这些蛋白质的增加与每种 mRNA 的表达几乎没有相关性。 ER应激诱导剂处理影响每个全长成熟形式，并且在wt和TXNDC11缺陷细胞中观察到的每个全长形式的差异较小。结论本研究证明TXNDC11是受IRE1-sXBP1通路调节的ER应激诱导基因。此外，TXNDC11 还参与 ATF6α、CREB3 和 CREB3L2 蛋白表达的调节，尽管对这些蛋白稳定性的贡献差异很大。因此，其进一步表征将为理解内质网生理学和病理学中的蛋白质稳态提供新的见解。
BackgroundsThe endoplasmic reticulum (ER) is a crucial organelle that regulates both the folding, modification and transport of many proteins and senses certain stimuli inside and outside of cells. ER-associated degradation (ERAD), including SEL1L is a crucial mechanism to maintain homeostasis. In this study, we performed comparative proteome analysis in wild-type (wt) and SEL1L-deficient cells.Methods and resultsWe found constitutively high expression of thioredoxin domain-containing protein 11 (TXNDC11) mRNA and protein in our SEL1L-deficient HEK293 cells by RT-PCR and Western blot analysis. The TXNDC11 gene possesses a well-conserved unfolded protein response element (UPRE) around its transcription start site, and ER stress increased TXNDC11 mRNA and luciferase reporter activity via this putative UPRE in HEK293 cells. The amounts of TXNDC11 protein in wild-type and SEL1L-deficient cells with or without thapsigargin (Tg) treatment were parallel to their mRNAs in these cells, which was almost proportional to spliced XBP1 (sXBP1) mRNA expression. The establishment and characterization of TXNDC11-deficient HEK293 cells revealed that the expression of three different ER resident stress sensors, ATF6α, CREB3 and CREB3L2, is regulated by TXNDC11. The rate of disappearance of the three proteins by CHX treatment in wt cells was remarkably different, and the full-length CREB3L2 protein was almost completely degraded within 15 min after CHX treatment. TXNDC11 deficiency increased the expression of each full-length form under resting conditions and delayed their disappearance by CHX treatment. Interestingly, the degree of increase in full-length CREB3/CREB3L2 by TXNDC11 deficiency was apparently higher than that in full-length ATF6α. The increase in these proteins by TXNDC11 deficiency was hardly correlated with the expression of each mRNA. Treatment with ER stress inducers influenced each full-length mature form, and the difference in each full-length form observed in wt and TXNDC11-deficient cells was smaller.ConclusionThis study demonstrated that TXNDC11 is an ER stress-inducible gene regulated by the IRE1-sXBP1 pathway. In addition, TXNDC11 is involved in the regulation of ATF6α, CREB3 and CREB3L2 protein expression, although the contribution to the stability of these proteins is quite variable. Therefore, its further characterization will provide new insights for understanding protein homeostasis in ER physiology and pathology.