Abstract：
Previously, we found by constructing various luciferase reporters that a well-conserved ATF6-binding element in the CRELD2 promoter is activated by transient ATF6 overexpression. In this study, we established ATF6-deficient and ATF4-deficient cell lines to analyze CRELD2 mRNA and protein expression together with that of other ER stress-inducible factors. Our results showed that ATF6 deficiency markedly suppressed tunicamycin (Tm)-induced expression of unglycosylated CRELD2. This reduction reflected a decrease in the CRELD2 transcription level. On the other hand, a putative ATF4-binding site in the mouse CRELD2 promoter did not respond to Tm stimulation, but ATF4 loss resulted in reductions in CRELD2 mRNA and protein expression, accompanied by a decrease in Tm-induced ATF6 expression. In contrast, transient suppression of GADD34, an ATF4 downstream factor, suppressed Tm-induced CRELD2 protein expression without a decrease in ATF6 protein expression. Furthermore, we investigated the association of CRELD2 with a well-known ERAD substrate, namely, an α1-antitripsin truncation mutant, NHK, by generating various CRELD2 and NHK constructs. Coimmunoprecipitation of these proteins was observed only when the cysteine in the CXXC motif on the N-terminal side of CRELD2 was replaced with alanine, and the interaction between the two was found to be disulfide bond-independent. Taken together, these findings indicate that CRELD2 expression is regulated by multiple factors via transcriptional and posttranscriptional mechanisms. In addition, the N-terminal structure of CRELD2, including the CXXC motif, was suggested to play a role in the association of the target proteins. In the future, the identification and characterization of factors interacting with CRELD2 will be useful for understanding protein homeostasis under various ER stress conditions.
摘要：
以前,通过构建各种荧光素酶报告基因，我们发现CRELD2启动子中一个保守的ATF6结合元件被瞬时ATF6过表达激活。在这项研究中,我们建立了ATF6缺陷型和ATF4缺陷型细胞系,以分析CRELD2mRNA和蛋白表达以及其他ER应激诱导因子的表达.我们的结果表明，ATF6缺乏症显着抑制了衣霉素（Tm）诱导的非糖基化CRELD2的表达。这种减少反映了CRELD2转录水平的降低。另一方面,小鼠CRELD2启动子中推定的ATF4结合位点对Tm刺激没有反应，但ATF4丢失导致CRELD2mRNA和蛋白表达减少，伴随着Tm诱导的ATF6表达的降低。相比之下,瞬态抑制GADD34，ATF4下游因子，抑制Tm诱导的CRELD2蛋白表达而不降低ATF6蛋白表达。此外，我们调查了CRELD2与众所周知的ERAD底物的关联，即,α1-抗曲别素截断突变体，NHK,通过生成各种CRELD2和NHK构造。只有当CRELD2的N端侧的CXXC基序中的半胱氨酸被丙氨酸取代时，才观察到这些蛋白质的免疫共沉淀。发现两者之间的相互作用是不依赖二硫键的。一起来看,这些发现表明CRELD2的表达通过转录和转录后机制受到多种因素的调节。此外,CRELD2的N端结构，包括CXXC基序，建议在靶蛋白的关联中发挥作用。在未来,与CRELD2相互作用的因子的鉴定和表征将有助于理解各种ER应激条件下的蛋白质稳态。
