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Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

基本信息

批准号：
7732889
负责人：
ETTORE APPELLA
金额：
$ 78.54万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7732889
关键词：
Acetylation Affect Affinity Alanine Alleles Antibody Affinity Apoptosis Arginine Binding Binding Sites Biochemical Biological Biological Assay CREB1 gene Cell Aging Cell Cycle Arrest Cell model Cells Cellular Assay Charge Chromatin Code Collaborations Compatible Complex Coupled Cyclic Peptides DNA Damage DNA Repair EP300 gene Enzymes Event Exposure to Gene Targeting Genes Genetic Transcription Genotoxic Stress HCT116 Cells Histones Human Human Development In Vitro Individual Ionizing radiation Knock-in Mouse Lead Link Lysine Malignant Neoplasms Mass Spectrum Analysis Mediation Mediator of activation protein Messenger RNA Methylation Missense Mutation Modification Molecular Mono-S Mus Mutant Strains Mice Mutate Mutation N-terminal Numbers PCAF gene Pathway Analysis Pathway interactions Pattern Phosphoric Monoester Hydrolases Phosphorylation Physiological Post-Transcriptional Regulation Post-Translational Modification Site Post-Translational Protein Processing Protein Dephosphorylation Protein Overexpression Protein p53 Proteins Proteomics Proto-Oncogenes Pyrroles Rate Recombinants Recruitment Activity Regulation Reporting Research Research Project Grants Resources Response Elements Role S Phase Sampling Screening procedure Series Serine Signal Pathway Signal Transduction Site Solutions Specificity Structure Substrate Specificity TP53 gene Testing Threonine Tissues Transactivation Transcript Transcription Coactivator Transcription Initiation Transcription Initiation Site Transferase Translation Initiation Translations Tumor Suppressor Proteins Untranslated Regions Wild Type Mouse Work base design functional group human FRAP1 protein in vivo inhibitor/antagonist kinase inhibitor knock-down mouse model mutant novel prevent pro-apoptotic protein promoter protein function protein phosphatase 2C protein protein interaction response scaffold small molecule thymocyte transcription factor tumor tumorigenesis ultraviolet

项目摘要

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 stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of post-translational modifications on p53 to better understand how they modulate p53 function. Our work on the transcriptional activating function of p53 has focused on its ability to recruit histone acetyl transferase co-activators such as CBP, p300 and PCAF to the promoters of specific target genes. We recently characterized the complex formed between the N-terminal transactivation domain (TAD) of p53 and the Taz2 domain of p300 and the effect of p53 phosphorylation on complex formation. We have determined the effect of various mono-, di-, and tri-phosphorylations on binding of p53(1-39) to Taz2, some of which significantly increased binding. Furthermore, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Mouse models containing missense mutations at sites of post-translational modification of p53, developed as part of a collaboration, continue to be a valuable resource for investigating the complex effects of single or multiple PTMs of p53 in a physiological setting. To elucidate functions of specific PTMs after DNA damage, knock-in mice were generated in which Ser18 (Ser15 in humans) was mutated to alanine in both alleles of endogenous p53, preventing phosphorylation. Additionally, knock-in mice were generated in which Lys317 (Lys320 in humans) was mutated to arginine, maintaining a positive charge but blocking PCAF acetylation at this site. Global proteomics analyses were performed using cleavable ICAT quantitative mass spectrometry to determine the change in the response of thymocytes to ionizing radiation (IR) in the mutant knock-in mice as compared with wild type (wt) mice. While many proteins were found to be significantly affected by IR in the wt thymocytes, there was a decrease in the number of affected proteins in the p53(S18A) thymocytes. Pro-apoptotic proteins that were increased in the wt after IR were not significantly affected in p53(S18A) mice, consistent with previous reports that thymocyte apoptosis is decreased in these mutant mice. When the effects of IR in the wt and p53(K317R) samples were compared, 46 proteins were found to be affected by the mutation (p less than 0.05), including proteins involved in apoptosis, transcription, and translation. Pathway analysis of the affected proteins suggests an increase in p53 activity in the p53(K317R) thymocytes, as well as a decrease in TNFalpha signaling. These results suggest that acetylation of Lys317 modulates the functions of p53 and influences the cross-talk between the DNA damage response and other signaling pathways. We have also studied the effects of p53 methylation on its function. Mediation of modular protein-protein interactions by lysine methylation of histone proteins regulates diverse chromatin signaling pathways. Lysines can be mono-, di- or tri- methylated, with the higher methylation states coupled to distinct functions by protein effectors that selectively recognize distinct lysine methylation events. Recently, lysine monomethylation of the tumor suppressor protein p53 has been shown to modulate its activity; however, the molecular mechanism that links monomethylated p53 to function is unclear. We have identified a novel p53 species dimethylated at Lys382 (p53K382me2) and shown that the tandem tudor domain (TD) of the DNA damage response mediator 53BP1 acts as an "effector" for this modification. We demonstrated that 53BP1(TD) preferentially recognizes p53K382me2 over other potential p53 and histone lysine methylation sites. Wip1 is a conserved PP2C phosphatase expressed at low levels in most tissues and transcriptionally induced after DNA damage in a p53-dependent manner. Wip1 substrates discovered thus far are p53, p38MAPK, UNG2, Chk1, Chk2 and ATM. Wip1 dephosphorylates serine and threonine residues within pTXpY and pT/SQ motifs, and we have recently characterized its substrate specificity using biochemical studies. As many of the known pT/SQ Wip1 substrates identified thus far are phosphorylated by ATM, we searched for motifs compatible with Wip1 specificity among known ATM targets to identify novel substrates. Recently, we found that Wip1 dephosphorylated Ser112 of 4E-BP1 in vitro , and interacted with proteins involved in the initiation of translation. In addition, Wip1 was able to negatively modulate eIF4E activity and the rate of cap-dependent translation both in cellular models and in vivo . The use of specific kinase inhibitors showed that the effect of Wip1 knock-down was independent of mTOR and p38MAPK, but required ATM activity. Therefore, the modulation of eIF4E activity by Wip1 may be occurring at two levels: by inhibiting ATM activity through dephosphorylation of Ser1981, and by directly dephosphorylating Ser112 of 4E-BP1. In cells containing wt p53, the levels of Wip1 mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. Using HCT116 p53+/+ and p53-/- cells, we have characterized a conserved p53 response element located in the 5′ untranslated region (UTR) of the Wip1 gene that is required for the p53-dependent induction of transcription from the human Wip1 promoter. CREB binding to the CRE contributes to the basal expression of Wip1 and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or IR, the abundance of transcripts with short 5′ UTRs increased in cells containing wt p53, indicating increased utilization of downstream transcription initiation sites. In these cells, exposure to UV resulted in increased Wip1 protein levels even when Wip1 mRNA levels remained constant, indicating post-transcriptional regulation of Wip1 protein levels. As described above, the Wip1 gene is amplified and overexpressed in some human tumors with wt p53, and our results support the hypothesis that Wip1 phosphatase is a candidate proto-oncogene that may promote tumorigenesis through inactivation of p53. We have identified several specific Wip1 inhibitors through a combination of rational design and a screening assay using recombinant Wip1. Using a pyrrole based scaffold, we have synthesized a series of small molecules that mimic the three-dimensional arrangement of the polar and hydrophobic functional groups of a previously identified cyclic-peptide inhibitor. Iterative optimization cycles of design, solution-phase synthesis and activity testing have led to an effective inhibitor of Wip1 that is selective for this phosphatase over related enzymes both in vitro and in cellular assays. We have used solution-based synthesis of the best inhibitor to produce quantities sufficient for in vivo studies and crystallographic analysis, which will facilitate further optimization of the binding selectivity and aff [summary truncated at 7800 characters]

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的翻译后修饰的生物学作用，以更好地了解它们如何调节p53的功能。我们对p53转录激活功能的研究主要集中在其招募组蛋白乙酰转移酶共激活因子（如CBP、p300和PCAF）到特定靶基因启动子的能力上。我们最近表征了p53的n端反活化结构域（TAD）和p300的Taz2结构域之间形成的复合物以及p53磷酸化对复合物形成的影响。我们已经确定了各种单磷酸化、二磷酸化和三磷酸化对p53（1-39）与Taz2结合的影响，其中一些磷酸化显著增加了p53与Taz2的结合。此外，我们在p53残基35-59中发现了Taz2的第二个结合位点。第二个位点与Taz2结合的亲和力与第一个位点相似，但结合不受磷酸化的影响。在p53翻译后修饰位点包含错义突变的小鼠模型，作为合作开发的一部分，继续成为研究单个或多个p53 ptm在生理环境中的复杂效应的宝贵资源。为了阐明DNA损伤后特异性PTMs的功能，我们产生了敲入小鼠，其中内源性p53的两个等位基因中的Ser18（人类中的Ser15）突变为丙氨酸，从而阻止了磷酸化。此外，产生敲入小鼠，其中Lys317（人类中的Lys320）突变为精氨酸，保持正电荷，但阻断PCAF在该位点的乙酰化。使用可切割ICAT定量质谱进行全局蛋白质组学分析，以确定与野生型（wt）小鼠相比，突变敲入小鼠胸腺细胞对电离辐射（IR）的反应变化。虽然在wt胸腺细胞中发现许多蛋白受到IR的显著影响，但p53（S18A）胸腺细胞中受IR影响的蛋白数量却有所减少。在p53（S18A）小鼠中，IR后wt中增加的促凋亡蛋白在p53（S18A）小鼠中未受到显著影响，这与先前报道的这些突变小鼠胸腺细胞凋亡减少一致。当比较IR对wt和p53（K317R）样本的影响时，发现46个蛋白受到突变的影响（p < 0.05），包括参与凋亡、转录和翻译的蛋白。受影响蛋白的通路分析表明p53（K317R）胸腺细胞中p53活性增加，以及TNFalpha信号传导减少。这些结果表明，Lys317的乙酰化可以调节p53的功能，并影响DNA损伤反应与其他信号通路之间的串扰。我们还研究了p53甲基化对其功能的影响。通过赖氨酸甲基化组蛋白介导模块化蛋白相互作用调节多种染色质信号通路。赖氨酸可以被单甲基化、二甲基化或三甲基化，较高的甲基化状态通过选择性识别不同赖氨酸甲基化事件的蛋白质效应物与不同的功能相结合。最近，肿瘤抑制蛋白p53的赖氨酸单甲基化已被证明可以调节其活性；然而，将单甲基化p53与功能联系起来的分子机制尚不清楚。我们已经确定了一个新的p53物种在Lys382位点二甲基化（p53K382me2），并表明DNA损伤反应介质53BP1的串联tudor结构域（TD）作为这种修饰的“效应器”。我们证明53BP1（TD）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。Wip1是一种保守的PP2C磷酸酶，在大多数组织中低水平表达，在DNA损伤后以p53依赖的方式转录诱导。目前发现的Wip1底物有p53、p38MAPK、UNG2、Chk1、Chk2和ATM。Wip1在pTXpY和pT/SQ基序中去磷酸化丝氨酸和苏氨酸残基，我们最近通过生化研究表征了其底物特异性。由于迄今为止鉴定的许多已知的pT/SQ Wip1底物被ATM磷酸化，我们在已知的ATM靶点中寻找与Wip1特异性相容的基元来鉴定新的底物。最近，我们发现Wip1在体外使4E-BP1的Ser112去磷酸化，并与参与翻译起始的蛋白相互作用。此外，在细胞模型和体内，Wip1都能够负向调节eIF4E活性和帽依赖翻译的速率。特异性激酶抑制剂的使用表明，Wip1敲除的作用与mTOR和p38MAPK无关，但需要ATM活性。因此，Wip1对eIF4E活性的调节可能发生在两个水平上：通过Ser1981的去磷酸化抑制ATM活性，以及通过直接去磷酸化4E-BP1的Ser112。在含有wt p53的细胞中，Wip1 mRNA和蛋白水平在暴露于基因毒性应激后升高，但p53调控的机制尚不清楚。利用HCT116 p53+/+和p53-/-细胞，我们鉴定了位于Wip1基因5&#8242；非翻译区（UTR）的一个保守的p53应答元件，该元件是人类Wip1启动子依赖p53诱导转录所必需的。CREB与CRE结合有助于Wip1的基础表达，并指导上游位点的转录起始。暴露于紫外线（UV）或IR后，含有wt p53的细胞中含有短5&#8242；utr的转录本的丰度增加，表明下游转录起始位点的利用增加。在这些细胞中，暴露于紫外线导致Wip1蛋白水平升高，即使Wip1 mRNA水平保持不变，这表明Wip1蛋白水平的转录后调控。如上所述，Wip1基因在一些携带wt p53的人类肿瘤中被扩增和过表达，我们的研究结果支持了Wip1磷酸酶是一种候选原癌基因的假设，该基因可能通过p53的失活促进肿瘤的发生。我们通过合理设计和重组Wip1筛选试验的结合，确定了几种特定的Wip1抑制剂。使用基于吡咯的支架，我们合成了一系列小分子，模拟了先前鉴定的环肽抑制剂的极性和疏水性官能团的三维排列。设计、液相合成和活性测试的迭代优化周期已经产生了一种有效的Wip1抑制剂，该抑制剂在体外和细胞试验中对该磷酸酶具有选择性，优于相关酶。我们已经使用基于溶液的最佳抑制剂合成方法来生产足够数量的体内研究和晶体学分析，这将有助于进一步优化结合选择性和aff[摘要截断为7800个字]