Synthesis and functional analyses of nuclear clusterin, a cell death protein
Synthesis and functional analyses of nuclear clusterin, a cell death protein
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DOI：
10.1074/jbc.m209233200
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发表时间：
2003-03-28
影响因子：
4.8
通讯作者：
Boothman, DA
中科院分区：
文献类型：
--
作者：
Leskov, KS;Klokov, DY;Boothman, DA
核聚集蛋白（Nuclear clusterin, nCLU）是一种电离辐射（IR）诱导蛋白，与Ku70结合，在MCF-7细胞中过表达可引发细胞凋亡。我们证明内源性nCLU合成是选择性剪接的产物。逆转录- pcr分析显示，含有第一个AUG并编码内质网靶向肽的外显子II被忽略。外显子I和III拼接在一起，将外显子III中的下游AUG作为第一个可用的翻译起始位点。这种较短的mRNA产生49 kda前体nCLU蛋白。Ku70的结合活性定位于nCLU的c端线圈结构域。nCLU的亮氨酸残基357、358和361是Ku70-nCLU相互作用所必需的。nCLU的N端和c端卷曲结构域相互作用，表明该蛋白可以二聚化或折叠。突变分析表明，c端NLS在nCLU中具有与n端NLS相同的功能。nCLU的c端卷曲结构域是Ku结合和凋亡所需的最小区域。MCF-7细胞凋亡细胞中GFP-nCLU在细胞核和细胞质中均有表达。在活细胞中发现GFP-nCLU的胞质聚集。这些结果表明，nCLU的失活前体存在于未辐照的MCF-7细胞的细胞质中，在辐照后易位到细胞核中，并诱导细胞凋亡。
Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis.