Comparative Analysis of CREB3 and CREB3L2 Protein Expression in HEK293 Cells.
Comparative Analysis of CREB3 and CREB3L2 Protein Expression in HEK293 Cells.
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DOI：
10.3390/ijms22052767
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发表时间：
2021-03-09
影响因子：
5.6
通讯作者：
Hirata Y
中科院分区：
文献类型：
--
作者：
Oh-Hashi K;Yamamoto A;Murase R;Hirata Y
我们使用药理学和基因组编辑方法在HEK293细胞中对两个ER驻留的CREB3家族蛋白CREB3和CREB3L2进行了比较分析，并确定了两者之间的一些差异。用灯盏花素A(BFA)和莫能菌素处理诱导了全长CREB3和CREB3L2的切割，但莫能菌素处理并没有显著降低全长CREB3蛋白的水平，而不是CREB3L2蛋白的水平。另一方面，衣霉素(TM)处理使全长CREB3L2蛋白的相对分子质量下降，但抑制了CREB3蛋白的表达。Thapsigargin(TG)只显著增加CREB3L2全长蛋白的表达，同时其裂解形式的表达水平略有增加。放线菌酮和MG132处理表明，内源CREB3和CREB3L2都是蛋白酶体底物。此外，α-甘露糖苷酶抑制剂Kifunensine显著增加了这两种全长形式的水平。与这些发现一致的是，缺乏SEL1L的细胞显示出全长CREB3和CREB3L2的表达增加；SEL1L是ER相关蛋白降解(ERAD)的关键成分；然而，放线菌酮处理比全长CREB3蛋白更迅速地下调SEL1L缺陷细胞的全长CREB3L2蛋白的表达。最后，我们研究了甘油三酯和BFA处理以及SEL1L缺失对几个CREB3和CREB3L2靶基因表达的诱导。综上所述，这项研究表明，内源性全长CREB3和CREB3L2都是ER相关蛋白降解的底物，但部分受到不同机制的调节，每一种机制都有助于产生不同于典型ER信号的独特细胞反应。
We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.
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影响因子：
4.3
作者：
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