Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

• 批准号: 8762998
• 负责人: ETTORE APPELLA
• 金额: $ 39.45万
• 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/8762998
• 关键词: Accounting; Acetylation; Affect; Affinity; Alanine; Alleles; Apoptosis; Binding; Binding Sites; Biological; C-terminal; Calcium Signaling; Cell Aging; Cell Cycle; Cell Cycle Arrest; Cells; Complement; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA Repair; DNA strand break; EP300 gene; Electrons; Energy Metabolism; Energy Metabolism Pathway; Engineering; Exhibits; Exposure to; Focal Adhesions; Frequencies; Future; Gene Expression; Gene Targeting; Genes; Genomics; Glioma; Goals; Human; In Vitro; Individual; International; Investigation; Ionizing radiation; Journals; Knock-in Mouse; Light; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Measures; Mediator of activation protein; Metabolism; Methods; Methylation; Missense Mutation; Modeling; Modification; Molecular Conformation; Mono-S; Mus; Mutagenesis; Mutation; N-terminal; Neoplasm Metastasis; Nucleotides; Oncogenes; Peptides; Phase; Phosphorylation; Phosphorylation Site; Physiological; Post-Translational Modification Site; Post-Translational Protein Processing; Protein Isoforms; Protein p53; Proteins; Publishing; Radiobiology; Radioisotopes; Regulation; Relative (related person); Repression; Research; Role; Sampling; Signal Pathway; Site; Slide; Solutions; Specificity; Stress; Structure; TP53 gene; Techniques; Tissues; Transactivation; Transcription Coactivator; Translations; Tumor Cell Mobility; Tumor Suppression; Tumor Suppressor Proteins; Work; alpha helix; cell type; embryonic stem cell; flexibility; histone modification; in vivo; insight; interest; malignant breast neoplasm; mouse model; overexpression; p53 Signaling Pathway; prevent; promoter; protein protein interaction; research study; response; self-renewal; stem cell differentiation; transcription factor; tumor; tumorigenesis

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 upon DNA damage by means of 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. Reciprocal negative regulation of p53 and Nanog maintains differentiation p53 supports the differentiation of embryonic stem cells (ESC) into differentiated states through suppression of NANOG, a gene required for ESC self-renewal. Previously, we showed that p53 Ser 315 phosphorylation was important for the suppression of Nanog expression during mouse ESC differentiation in a model containing a chimeric humanized p53 gene. p53 also suppresses dedifferentiation by maintaining suppression of NANOG in differentiated cells, a mechanism of tumor suppression demonstrated to be important in several human cancers, including gliomas and breast cancer. We investigated the roles of induced expression of Nanog in tumorigenesis and metastasis using an engineered mouse model. In a recent article published in Oncogene, we demonstrated that co-expression of Nanog and the oncogene Wnt in the mammary tissues of mice promoted tumorigenesis and metastasis. In this context, overexpression of Nanog activated the focal adhesion and calcium signaling pathways and suppressed the p53 signaling pathway, leading to increased tumor cell mobility, invasiveness and metastasis. Analysis of changes in gene expression between control tumors and tumors expressing high levels of Nanog revealed that the promoters of the most highly up-regulated genes exhibited the presence of Nanog transcription factor binding sites as well as the presence of both activating (H3K4me3) and repressive (H3K27me3) histone modifications. These results suggest that expression of Nanog in differentiated cells leads to inappropriate expression of genes with "poised" promoters that contribute to the metastasis of tumor cells. Global effects of p53 PTM Mouse models containing missense mutations at p53 PTM sites have been used to investigate the complex effects of p53 PTMs in a physiological setting. In these studies, we have primarily focused on knock-in mice containing mutation of Ser18 (human Ser15) to alanine in both alleles of endogenous p53, thereby preventing phosphorylation of this site. Our previous quantitative mass spectrometry studies of these mice demonstrated that the knock-in mutation affected proteins with roles in energy and metabolism pathways following ionizing radiation. As p53 has been shown to have important roles in regulation of metabolism and energy pathways, further investigation into modulation of these effects by phosphorylation is warranted. Thus, we are currently initiating studies to further understand the modulation of p53-dependent effects on metabolism in these knock-in mice. As a complement to mouse models, we are developing techniques to investigate global effects of p53 PTM in human cells. Recent advances in genomic editing provide practical methods for introducing specific modifications into genes in human cells. As protein translation accounts for a significant expenditure of energy by the cell, we are developing a sensitive method for analyzing protein translation in limited-availability samples. These methods will allow a more comprehensive investigation of the interrelationships between p53 PTMs and metabolism. 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 have determined the solution structure of a p53 TAD2 peptide in complex with Taz2. Upon binding to Taz2, p53 TAD2 forms a short alpha-helix, similar to the complex-dependent formation of a TAD1 alpha helix in the TAD1-Taz2 complex. Concomitant mutagenesis and binding studies have helped further characterize the complex. Comparison of the structures of the two complexes sheds light on how these two similar domains within p53 may function differently in co-activator recruitment after stress and suggests reasons for differences in transactivation between p53 and deltaNp53. In addition, as several new structures of p53 TAD2 complexes have been published, comparison of these structures with the TAD2-Taz2 complex will provide new understanding of the importance of flexibility in this domain for the formation of critical protein-protein interactions. Modulation of DNA binding by post-translational modification The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site. Thus, it provides a very sensitive measure of changes in the local conformation of the DNA. In a collaborative project with Victor Zhurkin (LCB) and Igor Panyutin (CC), we used radioprobing to study the conformation of DNA in complex with p53. This work, published in the International Journal of Radiation Biology, demonstrated that the most significant changes in the break frequency distributions were detected close to the center of the binding site, consistent with increased DNA twisting in this region as well as local DNA bending and sliding. Future studies will examine the effects of post-translational modification of the p53 DNA binding domain, including acetylation, on p53-induced DNA bending. Functional effects and interplay of p53 C-terminal modifications The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitinylation, sumoylation, and neddylation, that are primarily localized to the terminal thirty residues of the protein. We are interested in understanding the specific effects of individual site-specific modifications and the interplay between them. We have investigated the effects of mono- and dimethylation of p53 Lys382, a site that alternatively can be methylated, acetylated, or ubiquitinylated. Mono-methylation of p53 Lys382 results in repression of the activity of p53 as a transcription factor and we have continued to investigate the mechanism of repression. Dimethylation of p53 Lys382 is critical for the interaction of p53 with the tandem Tudor domain (TD) of the DNA damage response mediator 53BP1. We are currently exploring the role of additional modifications within the C-terminal regulatory domain that may combine with Lys382 dimethylation to further modulate the binding of p53 to the TD domain. Further experiments will provide insight into the interactions of 53BP1 with p53 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损伤时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。p53和Nanog的相互负调控维持分化p53通过抑制Nanog支持胚胎干细胞（ESC）向分化状态的分化，Nanog是ESC自我更新所需的基因。之前，我们在含有嵌合人源p53基因的小鼠ESC分化模型中发现p53 Ser 315磷酸化对于抑制Nanog表达很重要。p53还通过维持分化细胞中NANOG的抑制来抑制去分化，这一肿瘤抑制机制在包括胶质瘤和乳腺癌在内的几种人类癌症中被证明是重要的。我们利用工程小鼠模型研究了诱导Nanog表达在肿瘤发生和转移中的作用。在最近发表在Oncogene杂志上的一篇文章中，我们证明了Nanog和致癌基因Wnt在小鼠乳腺组织中的共同表达促进了肿瘤的发生和转移。在这种情况下，Nanog的过表达激活了局灶黏附和钙信号通路，抑制了p53信号通路，导致肿瘤细胞的移动性、侵袭性和转移性增加。对照肿瘤和高水平表达Nanog的肿瘤之间的基因表达变化分析显示，大多数高度上调基因的启动子显示出Nanog转录因子结合位点的存在以及激活（H3K4me3）和抑制（H3K27me3）组蛋白修饰的存在。这些结果表明，Nanog在分化细胞中的表达导致具有“平衡”启动子的基因的不适当表达，这些基因有助于肿瘤细胞的转移。在p53 PTM位点含有错义突变的小鼠模型已被用于研究p53 PTM在生理环境中的复杂作用。在这些研究中，我们主要关注内源性p53两个等位基因中含有Ser18（人类Ser15）到丙氨酸突变的敲入小鼠，从而阻止该位点的磷酸化。我们之前对这些小鼠的定量质谱研究表明，在电离辐射后，敲入突变影响了在能量和代谢途径中起作用的蛋白质。由于p53已被证明在代谢和能量通路的调节中具有重要作用，因此需要进一步研究磷酸化对这些作用的调节。因此，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。作为小鼠模型的补充，我们正在开发技术来研究p53 PTM在人类细胞中的整体效应。基因组编辑的最新进展为将特定修饰引入人类细胞中的基因提供了实用方法。由于蛋白质翻译在细胞中消耗了大量的能量，我们正在开发一种灵敏的方法来分析有限可用样品中的蛋白质翻译。这些方法将允许更全面地研究p53 ptm与代谢之间的相互关系。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，我们确定了p53 TAD2肽与Taz2复合物的溶液结构。在与Taz2结合后，p53 TAD2形成一个短的α -螺旋，类似于TAD1-Taz2复合体中TAD1 α -螺旋的复合体依赖性形成。伴随的诱变和结合研究有助于进一步表征该复合物。两种复合物结构的比较揭示了p53中这两个相似结构域在应激后共激活子募集中如何发挥不同的功能，并提出了p53和deltaNp53之间转激活差异的原因。此外，随着p53 TAD2复合物的几个新结构的发表，将这些结构与TAD2- taz2复合物进行比较，将使人们对该结构域的灵活性对关键蛋白-蛋白相互作用形成的重要性有新的认识。翻译后修饰对DNA结合的调节由俄歇电子发射放射性核素衰变产生的DNA链断裂的频率与DNA核苷酸离衰变点的距离成反比。因此，它提供了一种非常敏感的测量DNA局部构象变化的方法。在与Victor Zhurkin （LCB）和Igor Panyutin （CC）的合作项目中，我们使用放射探针研究了p53复合物中的DNA构象。这项发表在《国际辐射生物学杂志》（International Journal of Radiation Biology）上的研究表明，在靠近结合位点中心的地方检测到断裂频率分布的最显著变化，这与该区域DNA扭曲增加以及局部DNA弯曲和滑动一致。未来的研究将研究p53 DNA结合域的翻译后修饰（包括乙酰化）对p53诱导的DNA弯曲的影响。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、泛素化和类化修饰，这些修饰主要定位于蛋白质的末端30个残基。我们感兴趣的是了解单个位点特定修改的具体效果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。p53 Lys382的单甲基化导致p53作为转录因子的活性受到抑制，我们继续研究这种抑制的机制。p53 Lys382的二甲基化对于p53与DNA损伤反应介质53BP1的串联Tudor结构域（TD）的相互作用至关重要。我们目前正在探索c端调控域中的其他修饰的作用，这些修饰可能与Lys382二甲基化结合，进一步调节p53与TD结构域的结合。进一步的实验将深入了解53BP1与p53的相互作用，促进DNA损伤的修复。