Vojtesek B, Dolezalova H, Lauerova L, Svitakova M, Havlis P, Kovarik J, Midgley C A, Lane D P
Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic.
Oncogene. 1995 Jan 19;10(2):389-93.
The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has been determined. This core is flanked at the N-terminus by the transcriptional transactivation domain and at the C-terminus by sequences involved in the oligomerisation of the protein. Extensive immunochemical analysis of p53 has shown that dominant antigenic sites lie within these N- and C-terminal domains while few antibodies to the central core have been identified. One of these, PAb240, has been extensively characterised as its epitope is cryptic in the native DNA binding core structure but is exposed by denaturation. This epitope is also exposed on many p53 proteins that contain point mutations in the core domain suggesting that these mutations may have a common affect on the structure of the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synthetic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and distinguish mutant from wild-type conformations implying that the point mutations found in p53 in human tumours have widespread effects on the folding pattern of the DNA binding domain.
p53蛋白包含一个抗蛋白酶核心区域，该区域以序列特异性方式与DNA结合，其晶体结构已被确定。该核心区域在N端侧翼为转录反式激活结构域，在C端侧翼为参与蛋白质寡聚化的序列。对p53的广泛免疫化学分析表明，主要抗原位点位于这些N端和C端结构域内，而针对中央核心的抗体很少被鉴定出来。其中之一，PAb240，已被广泛表征，因为其表位在天然DNA结合核心结构中是隐蔽的，但通过变性而暴露。该表位在许多核心结构域中含有点突变的p53蛋白上也暴露，这表明这些突变可能对核心结构有共同影响。为了进一步研究这一点，我们针对p53上的新位点产生了几种新抗体，并使用合成肽绘制了它们的表位图谱。我们发现，针对核心中另外两个离散位点的抗体也能像PAb240一样识别隐蔽表位，并区分突变型和野生型构象，这意味着在人类肿瘤中p53中发现的点突变对DNA结合结构域的折叠模式有广泛影响。
