Analysis of the UPR, a transcriptional induction process coupled with intracellular signaling from the endoplasmic reticulum to the nucleus

到1999年，我们发现，我们分离的人碱性亮氨酸拉链蛋白ATF 6作为哺乳动物UPR特异性转录因子，组成型合成为位于内质网（ER）中的II型跨膜糖蛋白，并通过响应ER应激的蛋白水解激活。从2000年到2001年，我们进一步详细分析了ATF 6的各种性质，并获得了以下结果。(1)ATF 6的N-末端片段在ER应激诱导的蛋白水解后从ER重新定位到细胞核（通过间接免疫荧光分析可视化）。(2)哺乳动物UPR所必需的顺式作用ER应激反应元件（ERSE）的共有序列是CCAAT-N9-CCACG。尽管ATF 6本身不能结合ERSE，但当CCAAT部分结合到普遍存在的转录因子NF-Y时，ATF 6可以结合到ERSE的CCACG部分。(3)野生型和各种突变的ERSE样序列的转录活性与其结合ATF 6的能力密切相关。(4)300 μM浓度的丝氨酸蛋白酶抑制剂AEBSF可特异性阻断ER应激诱导的ATF 6蛋白水解和随后的BiP/GRP 78（ER中的主要分子伴侣）转录诱导。(5)对UPR和ATF 6靶基因的综合分析显示，大约一半的UPR靶基因直接受ATF 6调控，并且大多数ATF 6靶基因编码ER中的分子伴侣和折叠酶。(6)在具有家族性阿尔茨海默病相关的早老素基因突变的细胞中，ER应激诱导的ATF 6的激活被减轻，上述结果强烈地表明ATF 6在哺乳动物的UPR中起主要作用。

By 1999, we showed that the human basic leucine zipper protein ATF6 we isolated as a transcription factor specific to the mammalian UPR is constitutively synthesized as a type II transmembrane glycoprotein localized in the endoplasmic reticulum (ER) and activated by proteolysis in response to ER stress. From 2000 to 2001, we further analyzed various properties of ATF6 in more detail and obtained the following results.(1) The N-terminal fragment of ATF6 relocates from the ER to the nucleus upon ER stress-induced proteolysis (visualized by indirect immunofluorescence analysis). (2) The consensus sequence of the cis-acting ER stress response element (ERSE) necessary and sufficient for the mammalian UPR is CCAAT-N9-CCACG. Although ATF6 cannot bind to ERSE by itself, ATF6 can bind to the CCACG part of ERSE when the CCAAT part is bound to the ubiquitous transcription factor NF-Y. (3) Transcriptional activities of wild-type and various mutant ERSE-like sequences are correlated well with their abilities to bind ATF6. (4) ER stress-induced proteolysis of ATF6 and subsequent transcriptional induction of BiP/GRP78 (a major molecular chaperone in the ER) is specifically blocked by the serine protease inhibitor AEBSF at the concentration of 300 μM. (5) Comprehensive analysis of target genes of the UPR and ATF6 revealed that approximately half of UPR-target genes are directly regulated by ATF6 and that a majority of ATF6-target genes encodes molecular chaperones and folding enzymes in the ER. (6) In cells possessing Familial Alzheimer disease-linked mutation in the presenilin gene, ER stress-induced activation of ATF6 is mitigated.Above results strongly indicate that ATF6 plays a major role in the mammalian UPR.