Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

• 批准号: 7965056
• 负责人: ETTORE APPELLA
• 金额: $ 41.94万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965056
• 关键词: Ablation; Affect; Affinity; Alanine; Alleles; Apoptosis; Arginine; Aspartate; Binding; Binding Sites; Biological; Calorimetry; Cell Aging; Cell Cycle Arrest; Cells; Code; Collaborations; Complex; DNA Damage; DNA Repair; Dissociation; EP300 gene; Embryo; Fibroblasts; G1 Arrest; Gene Targeting; Generations; Genetic Transcription; Heating; Histones; Human; Hydrophobic Interactions; Induction of Apoptosis; Investigation; Ionizing radiation; Knock-in Mouse; Lead; Lysine; MAPK8 gene; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Metabolism; Methylation; Missense Mutation; Modeling; Modification; Molecular; Mutate; Mutation; N-terminal; PCAF gene; Pathway Analysis; Pathway interactions; Pattern; Peptides; Phosphorylation; Physiological; Polyribosomes; Post-Translational Modification Site; Post-Translational Protein Processing; Protein p53; Proteins; Regulation; Research; Resolution; Resources; Role; Signal Transduction; Site; Solutions; Structure; Surface; TP53 gene; Thermodynamics; Titrations; Transactivation; Transcriptional Activation; Transforming Growth Factor beta; Transgenic Mice; Transgenic Organisms; Translations; Tumor Suppressor Proteins; Ubiquitin-Conjugating Enzymes; Ubiquitination; Wild Type Mouse; Work; base; cell type; inhibitor/antagonist; inorganic phosphate; mouse model; mutant; novel; p300/CBP-Associated Factor; pressure; prevent; promoter; protein protein interaction; response; thymocyte; transcription factor

The human p53 protein is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. It is maintained at low levels in unstressed cells, but is stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of specific p53 PTMs to better understand how they modulate p53 function. We have identified a novel p53 species dimethylated at Lys382 (p53K382me2) and have shown that the tandem tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this modification. We found that the 53BP1 tudor domain preferentially recognizes p53K382me2 over other potential p53 and histone lysine methylation sites. p53K382me2 levels increase following DNA damage and this modificationfacilitates interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. The molecular mechanism of the nonhistone methyllysine recognition by the 53BP1 Tudor domain remained unknown. Recently, we obtained a 1.6 resolution crystal structure of the tandem Tudor domain of human 53BP1 in complex with a p53K382me2 peptide. In the complex, dimethylated Lys382 is bound in a cage formed by four aromatic residues and an aspartate of 53BP1. These results provide the basis for deciphering the role of this interaction in regulation of p53 and 53BP1 functions. Our work on the transcriptional activating function of p53 has focused in part on the recruitment of coactivators, such as CBP, p300 and PCAF, to the promoters of specific target genes. We have recently characterized the binding of the N-terminal transactivation domain of p53 to the Taz2 domain of the acetyltransferase p300. We used binding, thermodynamic, and structural studies to better understand the determinants of the interaction of the Taz2 domain with p531-40. We found that the full TAD1 is required for maximal binding and solved the NMR solution structure of p531-40 bound to Taz2. In the complex, p531-40 forms a short α-helix and interacts with Taz2 through an extended surface stabilized primarily by hydrophobic contacts. Phosphorylation of p53 at Ser15 or Thr18 increased its affinity for Taz2, possibly due to interaction of the phosphates with neighboring arginine residues in Taz2 and altered hydrophobic interactions. Furthermore, a second binding site for Taz2 was identified in p5335-59. The second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Further investigation of Taz2 binding to these two sites by isothermal titration calorimetry indicated that upon complex formation the change in heat capacity at constant pressure, ΔCp, was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of ΔCp for Taz2 binding to the p531-40 compared to the p5335-59 suggested that the importance of nonpolar and polar interactions differs between the two sites. Phosphorylation of p53 at Thr81 also mediates p53 protein-protein interaction, in this case, the interaction of p53 with Ubc13. Previous work had demonstrated that Ubc13, an E2 ubiquitin-conjugating enzyme, regulates p53 localization and transcriptional activity. We recently have demonstrated that association of p53 and Ubc13 on polysomes requires ongoing translation and results in p53 ubiquitination that interferes with its tetramerization. JNK phosphorylation of p53 at Thr81 results in dissociation of the Ubc13-p53 complex, which in turn leads to p53 multimerization and transcriptional activation. Inhibition of JNK activity or expression of a nonphosphorylatable mutant of p53 maintained an Ubc13-p53 complex that inhibits p53 multimerization. Our findings reveal that the regulation of p53 multimerization requires the action of JNK and Ubc13 on polysome-bound p53. Post-translational modification of p53 regulates the interactions with its negative regulators Mdm2 and MdmX; phosphorylation of p53 at Thr18 and Ser20 disrupts these interactions, allowing p53 to be stabilized and activated. To better understand the functions of these overlapping regulators of p53, we have used transgenic mice containing human p53 in place of the endogenous protein. In this model, p53 activity was reduced compared with wild type mice. We have recently observed that either the use of an Mdm2-p53 binding inhibitor, nutlin-3a, or the knockdown of Mdm2 partially restored p53-mediated apoptosis and G1 arrest in transgenic mouse embryo fibroblasts. Ablation of MdmX in combination with nutlin-3a treatment of the transgenic cells resulted in induction of apoptosis and cell cycle arrest comparable to wild type cells. Thus, the studies in the transgenic mouse model demonstrate the importance of inhibition of both Mdm2 and MdmX for activation of p53. Mouse models containing missense mutations at PTM sites of p53, developed in collaboration, continue to be a valuable resource for investigating the complex global effects of single or multiple PTMs of p53 in a physiological setting. Knock-in mice have been generated in which Ser18 (Ser15 in humans) was mutated to alanine in both alleles of endogenous p53, preventing phosphorylation. To better understand the role of this modification in the response of p53 to DNA damage, quantitative mass spectrometry was used to determine the global effects of ionizing radiation (IR) on the levels of proteins in thymocytes of the p53S18A knock-in mice and resulted in the identification of 73 proteins that were differently affected (p<0.05). The p53S18A mutation has widespread effects on specific protein levels following IR, affecting the levels of proteins involved in apoptosis, transcription, translation, and metabolism. Pathway analysis of the differently regulated proteins suggests that p53 activity and TGF-beta signaling are decreased in the p53S18A thymocytes. These results suggest that phosphorylation of Ser18 modulates both transcriptional and non-transcriptional functions of p53, influencing signaling of different pathways in the cell.

人类 p53 蛋白是一种同源四聚体、序列特异性转录因子，在细胞凋亡、细胞周期停滞、细胞衰老和 DNA 修复中发挥着至关重要的作用。它在未受应激的细胞中维持在较低水平，但在 DNA 损伤后通过广泛的翻译后修饰 (PTM) 稳定并激活。我们的研究重点是识别和探索特定 p53 PTM 的生物学作用，以更好地了解它们如何调节 p53 功能。我们已经鉴定出一种在 Lys382 位点二甲基化的新型 p53 物种 (p53K382me2)，并表明 DNA 损伤反应介质 53BP1 的串联 tudor 结构域充当这种修饰的“效应子”。我们发现 53BP1 tudor 结构域优先识别 p53K382me2，而不是其他潜在的 p53 和组蛋白赖氨酸甲基化位点。 DNA 损伤后 p53K382me2 水平增加，这种修饰促进 p53 和 53BP1 之间的相互作用。 p53K382me2 的产生促进了 DNA 损伤时 p53 蛋白的积累，而 p53 水平的增加需要 53BP1。 53BP1 Tudor 结构域识别非组蛋白甲基赖氨酸的分子机制仍不清楚。最近，我们获得了人 53BP1 串联 Tudor 结构域与 p53K382me2 肽复合物的 1.6 分辨率晶体结构。在该复合物中，二甲基化的 Lys382 结合在由四个芳香族残基和 53BP1 的天冬氨酸形成的笼中。这些结果为破译这种相互作用在 p53 和 53BP1 功能调节中的作用提供了基础。我们对 p53 转录激活功能的研究部分集中在将 CBP、p300 和 PCAF 等共激活因子招募到特定靶基因的启动子上。我们最近表征了 p53 的 N 端反式激活结构域与乙酰转移酶 p300 的 Taz2 结构域的结合。我们利用结合、热力学和结构研究来更好地了解 Taz2 结构域与 p531-40 相互作用的决定因素。我们发现完整的 TAD1 是最大结合所必需的，并解析了 p531-40 与 Taz2 结合的 NMR 溶液结构。在复合物中，p531-40 形成短的 β-螺旋，并通过主要通过疏水接触稳定的扩展表面与 Taz2 相互作用。 p53 在 Ser15 或 Thr18 处的磷酸化增加了其对 Taz2 的亲和力，这可能是由于磷酸盐与 Taz2 中邻近的精氨酸残基的相互作用并改变了疏水相互作用。此外，在 p5335-59 中鉴定出了 Taz2 的第二个结合位点。第二个位点以与第一个位点相似的亲和力结合 Taz2，但这种结合不受磷酸化的影响。通过等温滴定量热法对 Taz2 与这两个位点的结合进行进一步研究表明，在复合物形成后，恒压下热容的变化，&#916;Cp，对这两个位点都是负的，这表明疏水相互作用的重要性。然而，与 p5335-59 相比，Taz2 与 p531-40 结合的 &#916;Cp 负值更大，表明两个位点之间非极性和极性相互作用的重要性不同。 p53 Thr81 处的磷酸化也介导 p53 蛋白质-蛋白质相互作用，在本例中是 p53 与 Ubc13 的相互作用。之前的研究表明，Ubc13（一种 E2 泛素结合酶）可调节 p53 定位和转录活性。我们最近证明，p53 和 Ubc13 在多核糖体上的结合需要持续翻译，并导致 p53 泛素化，从而干扰其四聚化。 p53 Thr81 位点的 JNK 磷酸化导致 Ubc13-p53 复合物解离，进而导致 p53 多聚化和转录激活。 抑制 JNK 活性或 p53 不可磷酸化突变体的表达可维持抑制 p53 多聚化的 Ubc13-p53 复合物。我们的研究结果表明，p53 多聚化的调节需要 JNK 和 Ubc13 对多核糖体结合的 p53 的作用。 p53 的翻译后修饰调节与其负调节因子 Mdm2 和 MdmX 的相互作用； p53 Thr18 和 Ser20 处的磷酸化会破坏这些相互作用，从而使 p53 得以稳定和激活。为了更好地了解这些 p53 重叠调节因子的功能，我们使用含有人 p53 的转基因小鼠代替内源蛋白。在该模型中，与野生型小鼠相比，p53 活性降低。我们最近观察到，使用 Mdm2-p53 结合抑制剂 nutlin-3a 或敲低 Mdm2 可以部分恢复转基因小鼠胚胎成纤维细胞中 p53 介导的细胞凋亡和 G1 期停滞。与野生型细胞相比，MdmX 的消融结合 nutlin-3a 处理转基因细胞导致了细胞凋亡和细胞周期停滞的诱导。 因此，转基因小鼠模型的研究证明了抑制 Mdm2 和 MdmX 对于激活 p53 的重要性。合作开发的 p53 PTM 位点含有错义突变的小鼠模型仍然是研究生理环境中 p53 的单个或多个 PTM 的复杂全局影响的宝贵资源。 敲入小鼠的内源性 p53 的两个等位基因中的 S​​er18（人类的 Ser15）突变为丙氨酸，从而防止磷酸化。为了更好地了解这种修饰在 p53 对 DNA 损伤的反应中的作用，使用定量质谱法来确定电离辐射 (IR) 对 p53S18A 敲入小鼠胸腺细胞中蛋白质水平的整体影响，并鉴定出 73 种受到不同影响的蛋白质 (p<0.05)。 p53S18A 突变对 IR 后的特定蛋白质水平具有广泛影响，影响与细胞凋亡、转录、翻译和代谢相关的蛋白质水平。对不同调节蛋白的通路分析表明 p53S18A 胸腺细胞中 p53 活性和 TGF-β 信号传导降低。这些结果表明 Ser18 的磷酸化调节 p53 的转录和非转录功能，影响细胞中不同途径的信号传导。