Oikawa Yu, Matsuda Eishou, Nishii Tomonori, Ishida Yasumasa, Kawaichi Masashi
Division of Gene Function in Animals, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
J Biol Chem. 2008 May 23;283(21):14242-7. doi: 10.1074/jbc.M802257200. Epub 2008 Mar 28.
We previously identified and characterized a murine BTB domain-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by small interfering RNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/propidium iodide labeling, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53(-/-) mouse embryonic fibroblast cells also activated caspase-3 and cleavage of poly(ADP-ribose) polymerase, indicating that CIBZ-associated apoptosis is mediated by a p53-independent pathway; however, because both common and distinct targets are regulated by CIBZ- and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3 and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.
我们之前鉴定并表征了一种含BTB结构域的小鼠蛋白CIBZ（人类中的ZBTB38），它与CtBP相互作用并结合甲基化的CpG。然而，其生理功能仍不清楚。据报道，由于CtBP参与了不依赖p53的程序性细胞死亡，我们在此研究CIBZ是否与细胞凋亡相关。我们发现CIBZ在增殖的C2C12细胞中高表达，但在血清饥饿诱导细胞凋亡时其表达水平下降。用小干扰RNA在C2C12细胞中敲低CIBZ可诱导细胞凋亡，这通过膜联蛋白V/碘化丙啶标记增加、caspase-3激活以及聚（ADP-核糖）聚合酶的裂解来确定。CIBZ抑制还激活了caspase-7和caspase-9，表明与CIBZ相关的细胞凋亡通过线粒体途径发生。值得注意的是，在p53基因敲除的小鼠胚胎成纤维细胞中敲低CIBZ也激活了caspase-3和聚（ADP-核糖）聚合酶的裂解，表明与CIBZ相关的细胞凋亡是由不依赖p53的途径介导的；然而，由于共同和不同的靶点都受与CIBZ和CtBP相关的细胞凋亡调控，我们得出结论，涉及不止一条途径。最后，通过诱变和体外caspase裂解试验，我们表明CIBZ是caspase-3的一种新型底物并鉴定出两个caspase-3识别位点。这些发现共同表明，CIBZ通过参与小鼠细胞凋亡的负调控发挥重要作用。
