BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner
BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner
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DOI：
10.1074/jbc.m400159200
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发表时间：
2004-06-18
影响因子：
4.8
通讯作者：
Kasid, U
中科院分区：
文献类型：
--
作者：
Broustas, CG;Gokhale, PC;Kasid, U
我们在此报告了位于人类染色体11q24.1上的一个新的无内含子基因BRCC2的结构-功能特征。BRCC2开放阅读框（327 bp）编码一个类似于12 kda的蛋白（108个氨基酸（aa）），主要位于细胞质中，在线粒体中较少。在细胞质和线粒体中也发现了BRCC2 cDNA的异位表达。BRCC2的外源表达在三种不同细胞系中引起凋亡细胞死亡，其证据是染色质凝聚增强、DNA断裂或亚g(1)期细胞数量增加。在人前列腺癌细胞（PC-3）中，抗凋亡分子Bcl-X-L的共表达有效地阻断了brcc2诱导的DNA断裂。在广谱caspase抑制剂Z-VAD-fmk存在下，将BRCC2 cDNA瞬时转染PC-3细胞（100 muM, 24 h），可消除DNA断裂。BRCC2的表达与caspase-3和caspase-9的激活一致。缺乏bh3样结构域（5 - 12aa， LPIEGQEI）的BRCC2 n端缺失突变体（10.2 kDa, delta1 - 16aa）或含有突变体bh3样结构域（亮氨酸5 ->谷氨酸）的BRCC2不能诱导细胞凋亡，而c端缺失突变体（6.8 kDa, delta62 - 108aa）保留了与全长BRCC2相当的细胞凋亡活性。最后，用阿霉素或过氧化氢（H2O2）处理HeLa细胞，导致线粒体（重膜）内源性BRCC2水平升高(阿霉素（100 ng/ml）， 5 h，类似于2倍；H2O2 (200mum)， 2小时，类似于2倍)。这些发现表明BRCC2作为促凋亡分子发挥作用，并提示BRCC2诱导了caspase依赖的线粒体细胞死亡途径。
We report here the structure-functional characterization of a novel intronless gene, BRCC2, located on human chromosome 11q24.1. BRCC2 open reading frame (327 bp) codes for an similar to12-kDa protein (108 amino acids (aa)) localized predominantly in the cytosol and to a lesser extent in the mitochondria. Ectopic expression of BRCC2 cDNA also was found in both the cytosol and mitochondria. Exogenous expression of BRCC2 caused apoptotic cell death in three different cell lines as evidenced by enhanced chromatin condensation, DNA fragmentation, or an enhanced number of cells in the sub-G(1) phase. In human prostate cancer cells (PC-3), BRCC2-induced DNA fragmentation was blocked efficiently by coexpression of the anti-apoptotic molecule, Bcl-X-L. Transient transfection of BRCC2 cDNA into PC-3 cells in the presence of a broad-range caspase inhibitor, Z-VAD-fmk (100 muM, 24 h), abrogated DNA fragmentation. Consistently, BRCC2 expression correlated with the activation of caspase-3 and caspase-9. An N-terminal deletion mutant of BRCC2 (10.2 kDa, Delta1-16 aa) lacking a BH3-like domain (5 - 12 aa, LPIEGQEI) or BRCC2 containing a mutant BH3-like domain ( leucine 5 -->glutamate) failed to induce apoptosis, whereas a C-terminal deletion mutant (6.8 kDa, Delta62-108 aa) retained the apoptotic activity comparable to the full-length BRCC2. Finally, the treatment of HeLa cells with doxorubicin or hydrogen peroxide (H2O2) led to an increase in the mitochondrial ( heavy membrane) level of endogenous BRCC2 ( doxorubicin ( 100 ng/ml), 5 h, similar to2-fold; H2O2 (200 muM), 2 h, similar to2-fold). These findings demonstrate that BRCC2 functions as a proapoptotic molecule and suggest that BRCC2 induces a caspase-dependent mitochondrial pathway of cell death.