Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library
Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library
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DOI：
10.1074/jbc.m310815200
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发表时间：
2004-01-02
影响因子：
4.8
通讯作者：
Ishioka, C
中科院分区：
文献类型：
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作者：
Shiraishi, K;Kato, S;Ishioka, C
温度敏感（ts）突变已被用作遗传和分子工具来研究许多基因产物的功能。每个 ts 突变蛋白可能包含温度依赖性分子内机制，例如 ts 构象变化。为了确定控制蛋白质功能的关键 ts 结构元件，我们从由 2,314 个 p53 错义突变组成的综合突变文库中筛选了 ts p53 突变体，以了解其通过酿酒酵母中 p53 结合序列的序列特异性反应性。我们分离出 142 个 ts p53 突变体，其中包括 131 个未报道的 ts 突变体。这些突变体聚集在 DNA 结合结构域的 β 链中，特别是在蛋白质的两个 β 折叠之一中，以及 15 个残基（Thr(155)、Arg(158)、Met(160)、Ala(161)、Val(172)、His(214)、Ser(215)、Pro(223)、Thr(231)、Thr(253)、 Ile(254)、Thr(256)、Ser(269)、Glu(271) 和 Glu(285)) 是 ts 热点。在142个突变体中，有54个在人骨肉瘤Saos-2细胞中进行了进一步检查，并证实89%的突变体在哺乳动物细胞中也是ts。 ts 突变体代表了 p53 结合序列的不同 ts 反式活性和构象特异性抗 p53 抗体的不同表位表达模式。这些结果表明p53核心DNA结合域中的分子内β-折叠是控制蛋白质功能的关键结构元件，并为寻找能够挽救突变p53功能的分子机制提供了线索。
Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. These mutants clustered in beta-strands in the DNA-binding domain, particularly in one of the two beta-sheets of the protein, and 15 residues (Thr(155), Arg(158), Met(160), Ala(161), Val(172), His(214), Ser(215), Pro(223), Thr(231), Thr(253), Ile(254), Thr(256), Ser(269), Glu(271), and Glu(285)) were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.