Recombinant ATM protein complements the cellular A-T phenotype
Recombinant ATM protein complements the cellular A-T phenotype
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DOI：
10.1038/sj.onc.1201319
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发表时间：
1997-07-10
期刊：
影响因子：
8
通讯作者：
Shiloh, Y
中科院分区：
文献类型：
--
作者：
Ziv, Y;BarShira, A;Shiloh, Y
共济失调毛细血管扩张症 (A-T) 是一种常染色体隐性遗传疾病，其特征为神经退行性变、免疫缺陷、癌症易感性、基因组不稳定和辐射敏感性。 A-T 的细胞表型表明信号转导途径存在缺陷，涉及自由基损伤激活细胞周期检查点以及介导特定有丝分裂刺激传递的其他途径。 负责基因 ATM 的产物属于有助于维持维持的大型蛋白质家族。各种生物体中的基因组稳定性和细胞周期进展，稳定表达全长 ATM 蛋白的重组载体是对其功能分析的有价值的工具，我们使用载体和宿主的组合构建并克隆了 ATM 的重组全长开放阅读框，克服了该序列固有的不稳定性，使用杆状病毒载体在昆虫细胞中稳定表达重组 ATM，尽管水平较低，并使用附加型表达载体在人类 A-T 细胞中稳定表达，氨基末端添加到蛋白质中的FLAG表位允许通过免疫印迹、免疫沉淀和免疫染色高度特异性地检测重组分子，并使用免疫亲和力进行分离。与内源性ATM类似，重组蛋白主要位于细胞核中，细胞质中水平较低。A-T细胞中ATM的异位表达恢复了对电离辐射和拟放射药物新制癌菌素的正常敏感性，以及辐射后DNA合成的正常模式，这代表了S期检查点。这些观察结果表明重组的表位标记蛋白是有功能的，将已知的 A-T 错义突变 Glu2904Gly 引入该分子，导致该蛋白明显不稳定并且无法补充 A-T 表型。这些发现表明 A-T 细胞的生理缺陷特征是由于 ATM 蛋白的缺失造成的，并且可以通过该蛋白的异位表达来纠正这种缺陷。
Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability and radiation sensitivity, The cellular phenotype of A-T points to defects in signal transduction pathways involved in activation of cell cycle checkpoints by free radical damage, and other pathways that mediate the transmission of specific mitogenic stimuli, The product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycle progression in various organisms, A recombinant vector that stably expresses a full-length ATM protein is a valuable tool for its functional analysis, We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that overcame an inherent instability of this sequence, Recombinant ATM was stably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vector, An amino-terminal FLAG epitope added to the protein allowed highly specific detection of the recombinant molecule by immunoblotting, immunoprecipitation and immunostaining, and its isolation using immunoaffinity, Similar to endogenous ATM, the recombinant protein is located mainly in the nucleus, with low levels in the cytoplasm, Ectopic expression of ATM in A-T cells restored normal sensitivity to ionizing radiation and the radiomimetic drug neocarzinostatin, and a normal pattern of post-irradiation DNA synthesis, which represents an S-phase checkpoint. These observations indicate that the recombinant, epitope-tagged protein is functional, Introduction into this molecule of a known A-T missense mutation, Glu2904Gly, resulted in apparent instability of the protein and inability to complement the A-T phenotype, These findings indicate that the physiological defects characteristic of A-T cells result from the absence of the ATM protein, and that this deficiency can be corrected by ectopic expression of this protein.