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p53蛋白是一种同型序列特异性转录因子，在凋亡，细胞周期停滞，细胞衰老和DNA修复中具有至关重要的作用。它在无重理的细胞中保持低水平，但通过广泛的翻译后修饰（PTM）在DNA损伤后稳定和激活。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。我们已经确定了在Lys382（p53k382me2）处的一种新型的p53种类，并表明DNA损伤响应介体53BP1的串联tudor结构域可作为此修饰的“效应子”。我们发现，在其他潜在的p53和组蛋白赖氨酸甲基化位点上，53BP1都铎式结构域优先识别p53k382me2。 DNA损伤后p53k382me2水平升高，这种修饰是p53和53bp1之间的相互作用。 p53k382me2的产生促进了在DNA损伤时发生的p53蛋白的积累，p53水平的增加需要53BP1。 53BP1都铎王结构域的非组蛋白甲基透析素识别的分子机制仍然未知。最近，我们获得了与p53K382me2肽复合物中人类53bp1的串联帝堂结构域的1.6分辨率晶体结构。在复合物中，二甲基化的lys382与四个芳族残基形成的笼子结合，而天冬氨酸为53bp1。这些结果为破译这种相互作用在p53和53bp1函数调节中的作用提供了基础。我们在p53的转录激活功能方面的工作部分集中在募集的共激活因子（例如CBP，P300和PCAF）上，以供特定靶基因的启动子募集。我们最近表征了p53的N末端反变性结构域与乙酰基转移酶P300的TAZ2结构域的结合。我们使用结合，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。我们发现最大结合需要完整的TAD1并求解了与TAZ2结合的p531-40的NMR溶液结构。在复合体中，p531-40形成了短螺旋体，并通过主要通过疏水接触稳定的延伸表面与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以及疏水相互作用改变。此外，在p5335-59中确定了TAZ2的第二个结合位点。第二个位点结合的TAZ2具有与第一个位点相似的亲和力，但是结合不受磷酸化的影响。通过等温滴定热量法对TAZ2结合与这两个位点的结合表明，在复杂形成时，恒定压力下的热容量在恒定压力下的变化对于两个地点来说都是阴性的，这表明疏水相互作用的重要性。但是，与p5335-59相比，TAZ2与p531-40结合的＆＃916; cp的负值更为负值，这表明两个地点之间非极性和极性相互作用的重要性有所不同。 p53在THR81处的磷酸化还介导了p53蛋白 - 蛋白质相互作用，在这种情况下，p53与UBC13的相互作用。先前的工作表明，UBC13是E2泛素偶联酶，调节p53定位和转录活性。我们最近证明，p53和UBC13在多聚体上的关联需要持续的翻译，并导致p53泛素化，从而干扰其四聚化。 p53在THR81处的JNK磷酸化导致UBC13-p53复合物的解离，进而导致p53多次化和转录激活。 抑制JNK活性或表达p53的不可磷酸化突变体维持了抑制p53多聚化的UBC13-p53复合物。我们的发现表明，p53多聚化的调节需要JNK和UBC13对多聚合体结合p53的作用。 p53的翻译后修饰调节与其负调节剂MDM2和MDMX的相互作用； p53在Thr18和Ser20处的磷酸化破坏了这些相互作用，从而使p53稳定并激活。为了更好地理解这些重叠的调节剂的功能，我们已经使用了含有人p53的转基因小鼠代替内源性蛋白。在此模型中，与野生型小鼠相比，p53活性降低。我们最近观察到，在转基因小鼠胚胎成纤维细胞中使用MDM2-P53结合抑制剂NUTLIN-3A或MDM2的敲除部分恢复了P53介导的凋亡和G1停滞。 MDMX与Nutlin-3A的转基因细胞的处理，导致凋亡和细胞周期停滞诱导与野生型细胞相当。 因此，转基因小鼠模型中的研究证明了抑制MDM2和MDMX对p53激活的重要性。在协作中开发的P53 PTM位点中包含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的复杂全球效应的宝贵资源。 已经产生了敲入小鼠，其中Ser18（人类中的Ser15）在内源性p53等位基因中突变为丙氨酸，以防止磷酸化。为了更好地理解这种修饰在p53对DNA损伤反应中的作用，使用定量质谱法来确定电离辐射（IR）对p53S18A敲击小鼠胸腺细胞蛋白质水平的全球影响，并导致73个蛋白质的识别，这些蛋白质受到了不同影响的73个蛋白质（P <0.05）。 p53s18a突变对IR后特异性蛋白质水平产生广泛影响，影响参与凋亡，转录，翻译和代谢的蛋白质水平。对不同调节蛋白的途径分析表明，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的磷酸化调节了p53的转录和非转录功能，从而影响细胞中不同途径的信号传导。