Molecular mechanism of CHOP dependent cell death induced by extracellular stresses

内质网应激是由于基因突变或蛋白质异常修饰导致内质网内错误折叠或错折叠蛋白的积聚所致。近年来，内质网应激诱导的细胞凋亡被阐明是导致神经退行性疾病和糖尿病的原因。申请者们正在推进对转录因子CHOP的分析研究，这些转录因子是在内质网应激下诱导的，与细胞凋亡相关。在这里，我们已经证明了TRB3，一种果蝇激酶样分子Tribble的人类同源物，是由ER应激诱导的，其启动子是通过ER应激诱导的转录因子CHOP和ATF4激活的。TRB3蛋白抑制CHOP和ATF4的转录活性，提示CHOP(可能也包括ATF4)信号通路通过负反馈机制受到TRB3的严格调控。此外，TRB3的过表达增强了内质网应激诱导的细胞死亡，而通过siRNA方法敲除TRB3则减少了细胞死亡。我们有…进一步用免疫沉淀-Western印迹法观察了TRB3与CHOP的相关性。TRB3对CHOP转录活性的抑制作用与蛋白质降解无关。此外，TRB3的表达不影响CHOP的二聚体形成和DNA结合活性。然而，与CHOP相关的TRB3通过其转录结构域与p300结合域重叠。过表达TRB3可显著抑制p300-CHOP复合体的形成。这些结果表明，TRB3可能作为一种拮抗剂来取代CHOP的p300，并控制其转录效应。此外，ATF4的反式激活活性也被TRB3抑制，这可能是由于其稳定性的降低和p300与ATF4的解离。众所周知，一些细胞生存所必需的产物是ATF4的下游靶点，这表明TRB3下调ATF4可能对内质网应激导致的细胞凋亡有很大贡献。较少

The endoplasmic reticulum(ER) stress is induced by the accumulation of misfolding or malfolding proteins in ER caused by genetic mutation or abnormal modification of proteins. Recently, it is clarified that the ER stress-induced apoptosis cause the neurodegenerative diseases and the diabetes. Applicants are advancing the study of analysis on transcription factor CHOP to which are induced at the ER stress, and are related of apoptosis. Here we have shown that the TRB3, one of the human homologs of a Drosophila kinase like molecule Tribbles, is induced by ER stress and its promoter is activated via ER stress-induced transcription factors, CHOP and ATF4. TRB3 proteins suppress the transcriptional activities of CHOP and ATF4, suggesting CHOP(probably also ATF4)signaling is strictly regulated by TRB3 via a negative feedback mechanism.In addition, overexpression of TRB3 enhances the ER stress-induced cell death, while the knockdown of TRB3 by a siRNA method decreased the cell death. We have … More also observed the association between TRB3 and CHOP by the immunoprecipitaion-Western blot methods. Inhibitory effect of TRB3 on the transcriptional activity of CHOP is Independent of protein degradation. Moreover, the expression of TRB3 did not influence the dimer formation and DNA binding activities of CHOP. However, TRB3 associated with CHOP through its transcriptional domain and this domain is overlapped with a p300 binding domain. Overexpression of TRB3 remarkably inhibited the formation of p300-CHOP complex. These results suggest that TRB3 may function as an antagonist to replace the p300 from CHOP, and control its transcriptional effect. Moreover, transactivational activity of ATF4 is also suppressed by TRB3 probably by the decrease in its stability and by the dissociation of p300 from ATF4.It is well known that some products necessary for cell survival are the downstream targets of ATF4, suggesting that the downregulation of ATF4 by TRB3 may greatly contribute the apoptosis according to the ER stress. Less