A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.
A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.
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一种针对 p53 羧基末端多磷酸化构象表位的单克隆抗体。
DOI：
10.1016/s0167-4889(98)00087-1
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Ertl,HC
中科院分区：
文献类型：
--
作者：
OtvosJr,L;Hoffmann,R;Xiang,ZQ;O,I;Deng,H;Wysocka,M;Pease,AM;Rogers,ME;Blaszczyk-Thurin,M;Ertl,HC
编码肿瘤抑制蛋白p53的基因突变是在大约一半的人类肿瘤中发现的最常见的癌细胞分子改变。聚集在定义明确的热点上的突变改变了蛋白质的结构，从而影响了它与DNA结合的能力。翻译后修饰，主要是磷酸化，也可能影响p53如何与DNA结合或折叠成活性四聚体形式。然而，缺乏适当的生化标记来表征不同细胞类型和不同肿瘤进展阶段细胞的磷酸化状态，这阻碍了此类研究。为了产生一种敏感且磷酸化特异性的单克隆抗体（mAb），我们以双磷酸化的形式化学合成了人类p53的c -末端23个氨基酸片段。在H-2khaplotype小鼠中，携带Ser378和Ser392上磷酸基团的肽371-393与诱导转向的间隔物和肽31D（一种免疫优势的t辅助细胞表位）共同合成。脾细胞免疫并与骨髓瘤细胞融合后，获得了许多单抗，其中单抗p53-18是一种高度敏感的试剂。通过酶联免疫吸附试验，p53-18 （IgM同型的单抗）识别出在重组杆状病毒感染的昆虫细胞中表达的磷酸化p53，而在大肠杆菌中不表达p53。此外，来自昆虫细胞的小鼠p53可以用mAb p53-18免疫纯化。经过胰蛋白酶消化的质谱分析和液相色谱分析，证实在Ser375和Ser389处都存在磷酸基团。从相应的人蛋白片段中，mAb p53-18结合到Ser378和Ser392上磷酸化的免疫肽，但不能与未磷酸化的肽交叉反应，或者Ser378或Ser392上单独磷酸化的肽。然而，如果肽的构象稳定为α-螺旋构象，则可以恢复与未磷酸化肽的结合。人类血清（主要来自癌症患者）优先识别双磷酸化肽，而不是单磷酸化或未磷酸化的类似物，这一发现表明了p53多磷酸化c端的免疫原性。抗体p53-18是一种非常有用的生化标志物，可以检测不同组织中p53蛋白的低水平，是表征各种来源的p53蛋白c端磷酸化状态的关键工具。
Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371–393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2khaplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an α-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.
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蛋白激酶 C 的核定位。
影响因子：
3.9
作者：
Leach,KL;Raben,DM
通讯作者：
Raben,DM
猿猴病毒 40 的转化不涉及 p53 突变激活至致癌形式。
影响因子：
3.7
作者：
Lin,JY;Simmons,DT
通讯作者：
Simmons,DT
来自人p53基因产物羧基末端的特定序列在溶液中形成反平行四聚体。
DOI：
--
发表时间：
1994
影响因子：
11.1
作者：
H. Sakamoto;M. Lewis;H. Kodama;E. Appella;K. Sakaguchi
通讯作者：
K. Sakaguchi
酪蛋白激酶 II 和多胺可能在肾上腺皮质细胞对其营养激素的反应中相互作用。
DOI：
--
发表时间：
1991
期刊：
Biochemical and Biophysical Research Communications - BBRC
影响因子：
--
作者：
O. Filhol;P. Loue;C. Cochet;E. Chambaz
通讯作者：
E. Chambaz
合成磷酸肽异构体的反相高效液相色谱分离。
DOI：
10.1016/s0021-9673(01)89493-0
发表时间：
1990
期刊：
Journal of chromatography
影响因子：
--
作者：
OtvosJr,L;Tangoren,IA;Wroblewski,K;Hollosi,M;Lee,VM
通讯作者：
Lee,VM