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肿瘤抑制剂是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。它在无重大细胞中保持低水平，但通过广泛的翻译后修饰（PTM）稳定并激活DNA损伤。我们的研究重点是识别和探索p53 PTM的生物学作用，以更好地了解它们如何调节p53功能。 p53和Nanog的相互负调调节通过抑制Nanog（ESC自我更新所需的基因）来维持分化p53的分化p53。以前，我们表明p53 SER 315磷酸化对于抑制含有嵌合式人源化p53基因的模型中小鼠ESC分化过程中的纳米表达很重要。 p53还通过维持分化细胞中纳米的抑制来抑制去分化，这种抑制的机制在包括神经胶质瘤和乳腺癌在内的几种人类癌症中很重要。我们使用工程小鼠模型研究了纳米在肿瘤发生和转移中诱导的表达的作用。在最近发表在癌基因上的一篇文章中，我们证明了小鼠乳腺组织中纳米和癌基因Wnt的共表达促进了肿瘤发生和转移。在这种情况下，Nanog的过表达激活了局灶性粘附和钙信号通路，并抑制了p53信号通路，从而导致肿瘤细胞的迁移率增加，侵入性和转移。对对照肿瘤和表达高水平NANOG的肿瘤之间基因表达的变化的分析表明，最高度上调的基因的启动子表现出存在Nanog转录因子结合位点的存在以及激活（H3K4ME3）和抑制性（H3K27ME3）组酮组酮的启动（H3K4ME3）。这些结果表明，分化细胞中Nanog的表达会导致基因的不适当表达，从而有助于肿瘤细胞转移的“固定”启动子。 p53 PTM小鼠模型的全球效应含有p53 PTM位点的错义突变，用于研究p53 PTM在生理环境中的复杂效应。在这些研究中，我们主要集中在内源性p53等位基因中含有Ser18突变（人Ser15）突变的敲入小鼠，从而防止了该位点的磷酸化。我们先前对这些小鼠的定量质谱研究研究表明，敲入突变影响蛋白质在电离辐射后具有在能量和代谢途径中作用的蛋白质。由于已显示p53在代谢和能量途径的调节中具有重要作用，因此有必要进一步研究通过磷酸化对这些作用的调节。因此，我们目前正在启动研究，以进一步了解这些敲击小鼠中p53依赖性对代谢的影响的调节。作为对小鼠模型的补充，我们正在开发技术来研究p53 PTM在人类细胞中的全球效应。基因组编辑的最新进展提供了将特定修饰引入人类细胞基因的实用方法。随着蛋白质翻译构成了细胞的大量能量消耗，我们正在开发一种敏感方法，用于分析有限可用性样品中的蛋白质翻译。这些方法将允许对p53 PTM和代谢之间的相互关系进行更全面的研究。 p53 N末端磷酸化对p53，deltanp53的自然表达的同工型之一，缺乏p53的第一个反式激活结构域（TAD1），但确实包含第二个反式激活域（TAD2）。两种蛋白质的表达和稳定性受细胞类型，细胞周期阶段和暴露于各种应力的影响不同。 p53和deltanp53形成异驱动器，deltanp53的相对丰度影响p53的反式激活活性和靶基因特异性。我们对p53的TAD1和TAD2与转录共同p300的TAZ2结构域的结合表明，尽管与TAZ2绑定的两个域具有相等的亲和力，但TAD1的结合受p53磷酸化的影响，而TAD2的结合不受影响。为了更好地了解TAZ2与TAD1和TAD2的络合物之间的差异，我们确定了与TAZ2复合物中p53 TAD2肽的溶液结构。与TAZ2结合后，p53 TAD2形成一个短α-螺旋，类似于TAD1-TAZ2复合物中TAD1 Alpha螺旋的复杂依赖性形成。伴随诱变和结合研究有助于进一步表征复合物。两种配合物的结构的比较阐明了p53中的这两个相似域在压力后如何在共激活器募集中起不同，并提出了p53和deltanp53之间反式激活差异的原因。此外，随着p53 TAD2复合物的几种新结构已发表，将这些结构与TAD2-TAZ2复合物的比较将提供新的理解，从而有新的理解灵活性在该域中对临界蛋白质蛋白质相互作用的形成的重要性。通过翻译后修饰对DNA结合的调节e曲板发射放射性核素的衰减产生的DNA链断裂的频率与DNA核苷酸与衰减位点的距离成反比。因此，它提供了非常敏感的DNA局部构象变化的量度。在与Victor Zhurkin（LCB）和Igor Panyutin（CC）的合作项目中，我们使用放射线固定探针研究了与p53复杂的DNA的构象。这项发表在《国际放射线生物学杂志》上发表的这项工作表明，在接近结合位点的中心附近检测到断裂频率分布的最显着变化，这与该区域的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结合结构域的翻译后修饰（包括乙酰化）对p53诱导的DNA弯曲的影响。 p53 C末端修饰的功能效应和相互作用p53的C末端表现出各种各样的翻译后修饰，包括磷酸化，甲基化，乙酰化，乙酰化，泛素化，甲基硫代氨基化，舒张酰化和Neddylation，主要定位于末端thirirty protine protine protine protine protine protine protine protine protine nirtirty ot protein nirtirty ot an protein。我们有兴趣了解各个站点特异性修改及其之间的相互作用的特定效果。我们已经研究了p53 Lys382的单甲基化和二甲基化的作用，p53 lys382或可以甲基化，乙酰化或泛素化的位点。 p53 lys382的单甲基化导致p53作为转录因子的活性抑制，我们继续研究抑制的机理。 p53 lys382的二甲基化对于与DNA损伤响应介体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末端调节域内其他修改的作用，该域可能与LYS382二甲基化结合，以进一步调节p53与TD结构域的结合。进一步的实验将为53BP1与p53的相互作用提供洞察力，从而有助于修复DNA损伤。