Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

• 批准号: 8348885
• 负责人: ETTORE APPELLA
• 金额: $ 43.69万
• 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/8348885
• 关键词: Acetylation; Affect; Affinity; Alanine; Alleles; Apoptosis; Aspartate; Binding; Biological; C-terminal; Calorimetry; Cations; Cell Aging; Cell Cycle; Cell Cycle Arrest; Cells; Complex; DNA Damage; DNA Repair; EP300 gene; Energy Metabolism Pathway; Epitopes; Equilibrium; Exhibits; Exposure to; Gene Targeting; Goals; Heating; Histidine; Histones; Human; In Vitro; Ionizing radiation; Kinetics; Knock-in Mouse; Light; Lysine; Mass Spectrum Analysis; Measures; Mediator of activation protein; Metabolism; Methods; Methylation; Missense Mutation; Modification; Molecular Conformation; Mono-S; Mutagenesis; Mutation; N-terminal; Oxidative Phosphorylation; Pathway interactions; Peptides; Phase; Phosphorylation; Phosphorylation Site; Physiological; Post-Translational Modification Site; Post-Translational Protein Processing; Process; Protein Isoforms; Protein p53; Proteins; Protons; Regulation; Relative (related person); Research; Resolution; Role; Site; Solutions; Specificity; Stress; Structure; Titrations; Transactivation; Transcription Coactivator; Tromethamine; Tumor Suppressor Proteins; alpha helix; cell type; enthalpy; gene repression; in vivo; insight; interest; ionization; mouse model; mutant; prevent; pro-apoptotic protein; protein protein interaction; protonation; research study; response; thymocyte; transcription factor

The p53 tumor suppressor 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 stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 function. Global effects of p53 PTM We have used mouse models containing missense mutations at p53 PTM sites to investigate the complex effects of p53 PTMs in a physiological setting. Knock-in mice were generated containing mutation of Ser18 (Ser15 in humans) to alanine in both alleles of endogenous p53, thereby preventing phosphorylation of this site. Quantitative mass spectrometry analysis of wild type or p53S18A thymocytes was performed to investigate the role of this modification in the response of p53 to ionizing radiation (IR). The primary effect of the p53S18A mutation was a loss of wild type response to IR. Among those proteins that were differently affected were several pro-apoptotic proteins, which increased in protein level after IR in the wild type and were either unaffected by IR in the mutant or showed less increase than in the wild type. A second group of proteins that was differently affected by the mutation contains proteins with roles in energy and metabolism pathways. For example, two proteins that are critical for oxidative phosphorylation, a process that is promoted by p53, were both found to increase after IR in the wild type but were unaffected by IR in the mutant. The role of p53 in energy pathways is only recently becoming known, and these studies critically highlight new functions for p53 regulation by post-translational modification. We are currently initiating studies to further understand the modulation of p53-dependent effects on metabolism in these knock-in mice. Effects of p53 N-terminal phosphorylation on its protein-protein interactions One of the naturally expressed isoforms of p53, deltaNp53, lacks the first transactivation domain (TAD1) of p53 but does contain the second transactivation domain (TAD2). The expression and stability of the two proteins are affected differently by cell type, cell cycle phase and exposure to various stresses. p53 and deltaNp53 form heterotetramers and the relative abundance of deltaNp53 influences the transactivation activity and target gene specificity of p53. Our characterization of the binding of TAD1 and TAD2 of p53 to the Taz2 domain of the transcriptional coactivator p300 demonstrated that although the two domains bound to Taz2 with equal affinity, the binding of TAD1 was affected by p53 phosphorylations, whereas the binding of TAD2 was unaffected. To better understand the differences between the complexes of Taz2 with TAD1 and TAD2, we are determining the solution structure of a p53 TAD2 peptide in complex with Taz2. p53 TAD2 binds to a similar region on Taz2 and also forms a short alpha-helix upon binding, exposing hydrophobic residues to form the primary stabilizing interactions with Taz2. Additionally, mutagenesis experiments within p53 TAD2 suggest that, although we did not previously see an effect on the binding affinity when Thr55 was phosphorylated, mutation of this site to alanine did substantially alter the conformation of the complex. We will continue to investigate these findings using biophysical methods. Furthermore, comparison of the structures of the two complexes is anticipated to shed light on how these two similar domains within p53 may function differently in co-activator recruitment after stress. Moreover, it may help elucidate some of the differences in transactivation between p53 and deltaNp53. Functional effects and interplay of p53 C-terminal modifications The C-terminus of p53 exhibits diverse post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitinylation, sumoylation, and neddylation. We are interested in understanding the effects of these various site-specific modifications and the interplay between them. We have investigated the effects of mono- and dimethylation of p53 Lys382, a site that can be methylated, acetylated, or ubiquitinylated. We have continued to investigate the mechanism of p53 transcriptional repression by this modification. Following DNA damage, Lys382 becomes dimethylated, and we showed that this modification is critical for the interaction of p53 with the tandem Tudor domain (TD) of the DNA damage response mediator 53BP1. We obtained a 1.6 angstrum resolution crystal structure of the TD in complex with a p53 Lys382 dimethylated peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation-pi interactions in a cage formed by four aromatic residues and an aspartate of the TD. However, in the complex, most of the p53 residues were not well resolved, suggesting that the peptide may bind in more than one conformation. As a means of understanding the determinants of binding in addition to the dimethylated lysine, we have been using biophysical means to investigate the binding of the C-terminal regulatory domain to 53BP1. We examined the binding of p53 377-386 to 53BP1 by isothermal titration calorimetry and observed that although mutation of His380 or Lys381 did not significantly affect the affinity of the interaction, it did significantly affect the enthalpy of complex formation. As these experiments were performed at pH 7.5 using Tris buffer, which has a large heat of ionization, we hypothesized that the histidine protonation state could be important for the interaction of p53 with 53BP1, with the observed large negative deltaH being indicative of the release of a proton upon complex formation. We next used isothermal titration calorimetry to analyze the binding of p53377-386 Lys382me2 to 53BP1 at different pH values. We found that the affinity of p53377-386 Lys382me2 binding to 53BP1 was similar at pH 5.9, 7.5, and 8.5. Thus, these results indicate that the protonation state of the His380 does not affect the equilibrium binding constant. However, the protonation state may modulate the kinetics of p53 binding to 53BP1. We are currently pursuing studies to measure the kon and koff rates for p53377 386 Lys382me2 binding to 53BP1 to better understand the determinants of the interaction of the p53 C-terminal regulatory domain with the Tudor domain of 53BP1. These experiments will provide insight into the interactions of 53BP1 with p53 and histones that facilitate repair of DNA damage.

p53肿瘤抑制因子是一种同源四聚体、序列特异性转录因子，在细胞凋亡、细胞周期阻滞、细胞衰老和DNA修复中起着至关重要的作用。它在非应激细胞中维持在低水平，但在DNA损伤后通过广泛的翻译后修饰（PTM）稳定和激活。我们的研究重点是识别和探索p53 PTMs的生物学作用，以更好地了解它们如何调节p53的功能。我们使用含有p53 PTM位点错义突变的小鼠模型来研究p53 PTM在生理环境中的复杂作用。产生的敲入小鼠在内源性p53的两个等位基因中含有Ser18（人类是Ser15）到丙氨酸的突变，从而阻止了该位点的磷酸化。我们对野生型或p53S18A胸腺细胞进行了定量质谱分析，以研究这种修饰在p53对电离辐射（IR）的反应中的作用。p53S18A突变的主要影响是失去野生型对IR的反应。在这些受到不同影响的蛋白中，有几种促凋亡蛋白，在野生型中，它们在红外照射后的蛋白水平增加，而在突变体中，它们要么不受红外照射的影响，要么比野生型的增加少。受突变影响不同的第二组蛋白质包含在能量和代谢途径中起作用的蛋白质。例如，两种对氧化磷酸化至关重要的蛋白质，在野生型中都被发现在IR后增加，但在突变型中不受IR影响。氧化磷酸化是由p53促进的过程。p53在能量通路中的作用直到最近才为人所知，这些研究重点强调了p53通过翻译后修饰调控的新功能。我们目前正在开展研究，以进一步了解p53依赖性对这些敲入小鼠代谢的调节作用。p53 n端磷酸化对蛋白-蛋白相互作用的影响p53的自然表达亚型之一deltaNp53缺乏p53的第一反活化结构域（TAD1），但含有第二反活化结构域（TAD2）。这两种蛋白的表达和稳定性受到细胞类型、细胞周期阶段和暴露于各种胁迫的不同影响。p53和deltaNp53形成异源四聚体，deltaNp53的相对丰度影响p53的转激活活性和靶基因特异性。我们对p53的TAD1和TAD2与转录辅激活因子p300的Taz2结构域结合的表征表明，尽管这两个结构域以相同的亲和力与Taz2结合，但TAD1的结合受到p53磷酸化的影响，而TAD2的结合则不受影响。为了更好地了解Taz2与TAD1和TAD2复合物之间的差异，我们正在测定p53 TAD2肽与Taz2复合物的溶液结构。p53 TAD2与Taz2上的类似区域结合，并在结合时形成短的α -螺旋，暴露疏水残基与Taz2形成主要的稳定相互作用。此外，p53 TAD2的诱变实验表明，尽管我们之前没有看到Thr55磷酸化对结合亲和力的影响，但该位点对丙氨酸的突变确实大大改变了复合物的构象。我们将继续使用生物物理方法来研究这些发现。此外，对这两种复合物结构的比较有望揭示p53中这两个相似结构域在应激后共激活物募集中如何发挥不同的功能。此外，它可能有助于阐明p53和deltaNp53之间在转激活方面的一些差异。p53 c端修饰的功能作用和相互作用p53的c端表现出多种翻译后修饰，包括磷酸化、甲基化、乙酰化、泛素化、泛素化和类化。我们感兴趣的是了解这些不同的位点特异性修改的影响以及它们之间的相互作用。我们已经研究了p53 Lys382的单甲基化和二甲基化的影响，这个位点可以被甲基化、乙酰化或泛素化。我们继续通过这种修饰来研究p53转录抑制的机制。DNA损伤后，Lys382变成二甲基化，我们发现这种修饰对于p53与DNA损伤反应介质53BP1的串联都铎结构域（TD）的相互作用至关重要。我们获得了与p53 Lys382二甲基化肽复合物的TD的1.6埃分辨率晶体结构。在该复合体中，二甲基化的Lys382在由四个芳香残基和TD的天冬氨酸形成的笼中受到一系列疏水和阳离子- π相互作用的抑制。然而，在该复合体中，大多数p53残基没有被很好地分解，这表明肽可能以不止一种构象结合。除了二甲基化赖氨酸外，作为了解结合决定因素的一种手段，我们一直在使用生物物理方法研究c端调控域与53BP1的结合。我们用等温滴定量热法检测了p53 377-386与53BP1的结合，发现尽管His380或Lys381的突变对相互作用的亲和性没有显著影响，但对复合物形成的焓有显著影响。由于这些实验是在pH 7.5下使用Tris缓冲液进行的，该缓冲液具有较大的电离热，因此我们假设组氨酸质子化状态可能对p53与53BP1的相互作用很重要，观察到的大负δ tah表明在复合物形成时释放了一个质子。接下来，我们用等温滴定量热法分析了不同pH值下p53377-386 Lys382me2与53BP1的结合。我们发现p53377-386 Lys382me2与53BP1结合的亲和力在pH值5.9、7.5和8.5时相似。因此，这些结果表明His380的质子化状态不影响平衡结合常数。然而，质子化状态可能会调节p53与53BP1结合的动力学。我们目前正在进行研究，测量p53377 386 Lys382me2与53BP1结合的kon和koff率，以更好地了解p53 c -末端调控域与53BP1 Tudor结构域相互作用的决定因素。这些实验将深入了解53BP1与p53和组蛋白的相互作用，促进DNA损伤的修复。