Functional interactions of p53 with poly(ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: covalent poly(ADP-ribosyl)ation of p53 by exogenous PARP and noncovalent binding of p53 to the M(r) 85,000 proteolytic fragment.
Functional interactions of p53 with poly(ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: covalent poly(ADP-ribosyl)ation of p53 by exogenous PARP and noncovalent binding of p53 to the M(r) 85,000 proteolytic fragment.
复制标题
DOI：
--
复制
发表时间：
1998-11
期刊：
影响因子：
11.2
通讯作者：
Sunitha R. Kumari;H. Mendoza-Alvarez;R. Alvarez‐Gonzalez
中科院分区：
文献类型：
--
作者：
Sunitha R. Kumari;H. Mendoza-Alvarez;R. Alvarez‐Gonzalez
我们已经研究了p53和聚（ADP-核糖）聚合酶（PARP）之间的结构域特异性相互作用（E. C. 2.4.2.30）。通过将细胞暴露于50 μ M N-甲基-N '-硝基-N-亚硝基胍（MNNG）持续增加的时间长度来诱导细胞凋亡，并通过以下方式证实：（a）染色质的寡核小体片段化;（B）p53水平增加;和（c）PARP降解为特征性M（r）85，000（COOH末端催化结构域）和M（r）29，000（DNA结合结构域）肽片段。我们还免疫检测PARP特异性抗体获得的免疫沉淀物中的p53。然而，在MNNG处理的最初30分钟期间，完整的PARP与p53特异性抗体共免疫沉淀。60分钟后，只有COOH末端片段与p53共免疫沉淀，表明PARP通过其M（r）85，000催化结构域与p53非共价结合。因此，我们接下来检查p53作为聚（ADP-核糖基）化的共价靶。虽然p53在原位没有内源性聚（ADP-核糖基）化，但将细胞提取物与小牛胸腺全长PARP和[32 P] β NAD+孵育，导致其时间依赖性聚（ADP-核糖基）化。总之，我们的研究结果是一致的结论，PARP和p53的激活与非重叠的动力学在细胞凋亡。
We have examined the domain-specific interactions between p53 and poly(ADP-ribose)polymerase (PARP) (E.C. 2.4.2.30) in apoptotic HeLa cells. Apoptosis was induced by exposing cells to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for increasing lengths of time and was confirmed by: (a) oligonucleosomal fragmentation of chromatin; (b) increase in p53 levels; and (c) degradation of PARP into the characteristic M(r) 85,000 (COOH-terminal catalytic domain) and M(r) 29,000 (DNA-binding domain) peptide fragments. We also immunodetected p53 in immunoprecipitates obtained with a PARP-specific antibody. However, intact PARP coimmunoprecipitated with a p53-specific antibody during the initial 30 min of MNNG treatment. After 60 min, only the COOH-terminal fragment coimmunoprecipitated with p53, indicating that PARP noncovalently binds p53 via its M(r) 85,000 catalytic domain. Therefore, we next examined p53 as a covalent target for poly(ADP-ribosyl)ation. Although p53 was not endogenously poly (ADP-ribosyl)ated in situ, incubation of cell extracts with full-length PARP from calf thymus and [32P]beta NAD+ resulted in its time-dependent poly(ADP-ribosyl)ation. In summary, our results are consistent with the conclusion that PARP and p53 are activated with nonoverlapping kinetics during apoptosis.