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。我们的研究集中在识别和探索p53上翻译后修饰的生物学作用，以更好地了解它们如何调节p53功能。我们在p53的转录激活功能方面的工作集中在募集组蛋白乙酰基转移酶共激活剂（例如CBP，P300和PCAF）的能力上，以供特定靶基因的启动子。我们最近表征了p53的N末端反式激活结构域（TAD）与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，二和三磷酸化对p53（1-39）与TAZ2结合的影响，其中一些显着增加了结合。此外，我们确定了p53残基35-59中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。作为协作的一部分而开发的p53翻译后修饰部位的小鼠模型仍然是研究生理环境中p53的单个或多个PTM的复杂效应的宝贵资源。为了阐明DNA损伤后特定PTM的功能，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。此外，生成了敲入小鼠，其中将lys317（人类的Lys320）突变为精氨酸，保持正电荷，但在该部位阻止了PCAF乙酰化。与野生型（WT）小鼠相比，使用可裂解的ICAT定量质谱法进行了全局蛋白质组学分析，以确定突变型敲入小鼠中胸腺细胞对电离辐射（IR）的反应变化。尽管发现许多蛋白质受到WT胸腺细胞中IR的显着影响，但p53（S18A）胸腺细胞中受影响蛋白的数量减少。在p53（S18A）小鼠中，IR后WT中增加的凋亡蛋白没有显着影响，这与以前的报道一致，即这些突变小鼠中胸腺细胞凋亡降低。当比较WT和p53（K317R）样品中IR的作用时，发现46种蛋白质受到突变的影响（P小于0.05），包括参与凋亡，转录和翻译的蛋白质。对受影响蛋白的途径分析表明，p53（K317R）胸腺细胞的p53活性增加，以及TNFALPHA信号传导的降低。这些结果表明，Lys317的乙酰化调节p53的功能，并影响DNA损伤响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。通过组蛋白的赖氨酸甲基化对模块化蛋白 - 蛋白质相互作用的介导可调节多种染色质信号通路。赖氨酸可以是单，二或三甲基化的，较高的甲基化状态通过蛋白质效应子与不同的功能结合，这些蛋白质效应子选择性地识别不同的赖氨酸甲基化事件。最近，已证明肿瘤抑制蛋白p53的赖氨酸单甲基化可以调节其活性。但是，将单甲基化p53与功能联系起来的分子机制尚不清楚。我们已经确定了在Lys382（p53k382me2）处的一种新型p53物种，并表明DNA损伤响应介质53BP1的串联tudor结构域（TD）充当此修饰的“效应器”。我们证明了53BP1（TD）优先识别其他潜在p53和组蛋白赖氨酸甲基化位点的p53k382me2。 WIP1是一种保守的PP2C磷酸酶，在大多数组织中低水平表达，并以p53依赖性方式在DNA损伤后转录诱导。到目前为止发现的WIP1底物是p53，p38mapk，ung2，chk1，chk2和atm。 WIP1在PTXPY和PT/SQ基序中脱磷酸化丝氨酸和苏氨酸残基，我们最近使用生化研究表征了其底物的特异性。由于到目前为止确定的许多已知的PT/SQ WIP1底物通过ATM磷酸化，因此我们在已知的ATM目标之间搜索了与WIP1特异性兼容的基序，以识别新的底物。最近，我们发现在体外4E-BP1的WIP1去磷酸化的Ser112与参与翻译起始的蛋白质相互作用。此外，WIP1能够在细胞模型和体内负面调节EIF4E活性和cap依赖性翻译的速率。特定激酶抑制剂的使用表明，WIP1敲低的作用与MTOR和P38MAPK无关，但需要ATM活性。因此，WIP1对EIF4E活性的调节可能在两个级别上发生：通过通过SER1981的去磷酸化抑制ATM活性，以及通过直接将4E-BP1的SER112脱磷酸化的Ser112。在含有WT p53的细胞中，暴露于遗传毒性应激后的WIP1 mRNA和蛋白质的水平增加，但是p53调节的机制尚不清楚。使用HCT116 p53+/+和p53 - / - 细胞，我们表征了位于WIP1基因的5'未翻译区（UTR）中的保守p53响应元件，这是依赖人WIP1启动子转录所必需的。 CREB与CRE的结合有助于WIP1的基础表达，并指导上游位点的转录启动。暴露于紫外线（UV）或IR之后，含有WT p53的细胞中短短5'UTR的转录本的丰度表明，下游转录起始位点的利用率增加。在这些细胞中，即使WIP1 mRNA水平保持恒定，暴露于紫外线也会导致WIP1蛋白水平升高，表明WIP1蛋白水平的转录后调节。如上所述，WIP1基因在某些具有WT p53的人类肿瘤中被扩增和表达过表达，我们的结果支持以下假设：WIP1磷酸酶是一种候选原始癌基因，可以通过p53灭活症状。我们通过使用重组WIP1的合理设计和筛选测定的结合确定了几种特定的WIP1抑制剂。使用基于吡咯的支架，我们合成了一系列的小分子，这些分子模仿了先前鉴定的环肽抑制剂的极性和疏水官能团的三维布置。设计，溶液期合成和活性测试的迭代优化循环已导致WIP1的有效抑制剂，该抑制剂在体外和细胞测定中对这种磷酸酶的选择性优于相关酶。我们已经使用了基于溶液的最佳抑制剂的合成来产生足以用于体内研究和晶体学分析的数量，这将有助于进一步优化结合选择性和AFF [摘要以7800个字符截断]