Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

• 批准号: 10702285
• 负责人: ETTORE APPELLA
• 金额: $ 48.07万
• 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/10702285
• 关键词: Acetylation; Adenocarcinoma Cell; Affect; Amyloidosis; Apoptosis; Apoptotic; Attenuated; Binding; Biochemical; Biological; Biotin; C-terminal; CRISPR interference; CRISPR-mediated transcriptional activation; Cancer cell line; Catalytic Domain; Cell Cycle Arrest; Cell Line; Cells; Chemicals; Collaborations; Colon Carcinoma; Critical Pathways; DNA; DNA Damage; DNA Repair; Deubiquitinating Enzyme; EP300 gene; Esophageal Adenocarcinoma; Etoposide; Exhibits; Family; Genetic; Genetic Transcription; Goals; Growth; Heat-Shock Response; Homo; Hot Spot; Human; Hydroxylation; Hypoxia; Image; Lysine; MCF7 cell; Malignant neoplasm of ovary; Mass Spectrum Analysis; Metabolism; Methods; Methylation; Modeling; Modification; Molecular Conformation; Mutation; N-terminal; Nuclear Extract; Nuclear Proteins; Oncogenic; Outcome; Pathway interactions; Peptides; Pharmacology; Phosphorylation; Play; Point Mutation; Post-Translational Protein Processing; Proteins; Regulation; Reporting; Research; Role; Site; Small Interfering RNA; Stress; Sumoylation Pathway; TP53 gene; Transactivation; Transcription Coactivator; Transcriptional Activation; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; anticancer treatment; base; cancer stem cell; cofactor; druggable target; endoplasmic reticulum stress; experimental study; gain of function; high throughput screening; improved; in vivo; inhibitor; member; mutant; neuroblastoma cell; novel; peptidomimetics; preference; prevent; response; senescence; small molecule; transcription factor; tumor; tumor growth; tumorigenic; ultraviolet damage

The p53 tumor suppressor protein is a key component of the cellular response to stress. It is a homo-tetrameric, sequence-specific transcription factor activated by DNA damage, hypoxia, heat shock, and other types of stress and regulates DNA repair, cell cycle arrest, senescence, metabolism and apoptosis. It is maintained at low levels in unstressed cells but becomes 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 functions. The tandem N-terminal transactivation domains (TADs) of p53 are crucial for p53 activity as a transcription factor. The two subdomains, TAD1 (residues 1-40) and TAD2 (residues 35-59), interact with several domains of the transcriptional coactivator p300. However, the two subdomains can function independently of one another, suggesting the participation of distinguishing transcriptional cofactors in transcriptional activation by TAD1 and TAD2 in which interactions may be differentially regulated by p53 phosphorylation. To identify distinct interacting partners for TAD1 and TAD2, peptides comprising TAD1 (residues 9-33) or TAD2 (residues 35-59), with and without phosphorylation at Thr 18 or Ser 46, respectively, were synthesized and covalently attached to biotin at the N-termini. We used these peptides as a bait for pulldown of interacting proteins from nuclear extracts prepared from MCF7 cells treated with etoposide; mass spectrometry analysis was used to identify and quantitatively compare the interactors to discriminate between those preferentially interacting with the TAD1 or TAD2 subdomains. Our experiments using biological triplicate pulldowns have identified a list of potential interactors that show a preference for either unmodified or modified p53 in untreated cells or following etoposide treatment. In addition to known binding partners of p53 TAD1 and TAD2, we identified several new interactors. We have validated a few of the new interactors identified and are performing functional experiments to investigate the consequences of these interactions on p53 activity and stability. We have also expanded the experiments to determine whether these interactions also occur under other stress conditions known to activate p53, including ER stress and UV damage. Understanding the effects of these new interactors on p53 regulation will open new methods to increase p53 activity in tumors. The C-terminus of p53 exhibits a diverse array of PTMs, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, neddylation and hydroxylation that are primarily localized to the terminal thirty residues of the protein. We have shown that p53 can be both mono- and dimethylated on Lys382, with the former modification repressing p53 transcriptional activity and the latter promoting DNA repair, in addition to demonstrated acetylation and ubiquitination of the same site. SETD8 monomethylates p53 on lysine 382, attenuating p53 pro-apoptotic and growth arrest functions. Using a high-content imaging siRNA screen and a chemical screen, in a collaboration with Drs. Veschi and Thiele, we identified SETD8 as a suppressor of p53 activity in neuroblastoma cell lines. Genetic or pharmacological inhibition of SETD8 activity resulted in activation of the p53 wild-type pathway by decreasing p53K382me1. We have initiated a collaboration with Drs. Veschi and Stassi and recently showed that SETD8 is highly expressed in colon cancer stem cells (CSCs), with commensurate increased levels of p53K382me1. These two effects may play a synergistic role in the tumorigenic and metastatic capabilities of CSCs. We are currently using a high-throughput assay to identify novel inhibitors of SETD8 with an improved activity and tolerability in vivo. p53 point mutations have been reported to occur in approximately half of all human tumors, with marked over-representation of specific "hot-spot" residues. These mutations abolish the ability of p53 to function as a transcription factor and tumor suppressor. Moreover, many mutant forms of p53 have acquired novel oncogenic activities through gain-of-function mechanisms. p53 mutations generally either affect DNA contact or cause structural instability with partial unfolding and aggregate formation, similar to that seen in amyloid diseases. We are examining a small molecule, NSC59984, in esophageal adenocarcinoma cells and investigating its mechanism of action and effects on the oncometabolic profile. The findings will help elucidate pathways critical for preventing tumor growth by inhibiting gain-of-function mutant p53 activities and restoring wild-type p53 activity. A second means to target mutant p53 was reported by Padmanabhan et al. (Nat Commun. 9(1):1270 (2018)), showing that an inhibitor targeting deubiquitinase USP15 specifically results in p53-R175H degradation by disrupting the USP15-p53-R175H interaction. This leads to increased p53-R175H ubiquitination and degradation of the mutant protein. Based on these findings, we have chosen to explore other members of the deubiquitinating enzyme (DUB) family as potential modulators of mutant p53 protein stability. Using CRISPR-interference and CRISPR-activation screens with DUB-targeting sgRNAs in ovarian cancer cell lines of varying p53 status, we are identifying DUBs that contribute to mutant p53 stability. These lines encompass several p53 expression models, p53-null, wild-type and mutant p53-R175H, commonly seen in ovarian cancer. The DUBs that are identified will be further analyzed as druggable targets. The ability to selectively target DUBs will be crucial, since they take on several roles in the ubiquitin pathway, particularly by focusing on the N-terminal domains adjacent to the catalytic domain, which vary significantly among USPs. The goal is to develop small molecules and/or peptidomimetic compounds that disrupt the accumulation of mutant p53 and/or rescue p53 wildtype tumor suppressive function.

p53肿瘤抑制蛋白是细胞应激反应的关键组成部分。它是一种同源四聚体、序列特异性转录因子，可被DNA损伤、缺氧、热休克和其他类型的应激激活，调节DNA修复、细胞周期阻滞、衰老、代谢和凋亡。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）变得稳定和激活。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。作为一种转录因子，p53的串联n端反活化结构域（TADs）对p53的活性至关重要。这两个子结构域，TAD1（残基1-40）和TAD2（残基35-59），与转录辅激活子p300的几个结构域相互作用。然而，这两个亚结构域可以相互独立地发挥作用，这表明不同的转录辅助因子参与了TAD1和TAD2的转录激活，其中相互作用可能受到p53磷酸化的差异调节。为了确定TAD1和TAD2的不同的相互作用伙伴，合成了由TAD1（残基9-33）或TAD2（残基35-59）组成的肽，分别在Thr 18或Ser 46磷酸化和不磷酸化，并在n端共价连接到生物素上。我们使用这些肽作为诱饵，从经依托波苷处理的MCF7细胞制备的核提取物中提取相互作用蛋白；质谱分析用于鉴定和定量比较相互作用物，以区分优先与TAD1或TAD2子结构域相互作用的相互作用物。我们使用生物三重复下拉的实验已经确定了一系列潜在的相互作用物，这些相互作用物在未经处理的细胞或依托泊苷治疗后显示出对未修饰或修饰的p53的偏好。除了已知的p53 TAD1和TAD2结合伙伴外，我们还发现了几个新的相互作用物。我们已经验证了一些新的相互作用，并正在进行功能实验，以研究这些相互作用对p53活性和稳定性的影响。我们还扩展了实验，以确定这些相互作用是否也发生在其他已知的激活p53的应激条件下，包括内质网应激和紫外线损伤。了解这些新的相互作用物对p53调控的影响将开辟新的方法来增加肿瘤中p53的活性。p53的c端表现出多种PTMs，包括磷酸化、甲基化、乙酰化、泛素化、泛素化、类木化、类木化和羟基化，主要定位于蛋白质的末端30个残基。我们已经证明p53可以在Lys382上被单甲基化和二甲基化，前者的修饰抑制p53的转录活性，后者促进DNA修复，此外还证明了同一位点的乙酰化和泛素化。SETD8单甲基化赖氨酸382上的p53，减弱p53的促凋亡和生长抑制功能。使用高含量的成像siRNA屏幕和化学屏幕，在与博士合作。Veschi和Thiele发现SETD8是神经母细胞瘤细胞系中p53活性的抑制因子。遗传或药理学抑制SETD8活性可通过降低p53K382me1激活p53野生型通路。我们已经开始与博士合作。Veschi和Stassi以及最近的研究表明，SETD8在结肠癌干细胞（CSCs）中高度表达，p53K382me1的水平相应升高。这两种作用可能在CSCs的致瘤性和转移能力中发挥协同作用。我们目前正在使用一种高通量测定方法来鉴定具有改善活性和体内耐受性的SETD8的新型抑制剂。据报道，P53点突变发生在大约一半的人类肿瘤中，具有特定“热点”残基的明显过度代表。这些突变破坏了p53作为转录因子和肿瘤抑制因子的功能。此外，许多突变形式的p53通过功能获得机制获得了新的致癌活性。p53突变通常要么影响DNA接触，要么导致部分展开和聚集形成的结构不稳定，类似于淀粉样蛋白疾病。我们正在研究食管癌细胞中的一种小分子NSC59984，并研究其作用机制和对肿瘤代谢谱的影响。这些发现将有助于阐明通过抑制功能获得型突变p53活性和恢复野生型p53活性来防止肿瘤生长的关键途径。Padmanabhan等人报道了第二种靶向突变型p53的方法(Nat commin . 9(1):1270(2018))，表明靶向去泛素酶USP15的抑制剂通过破坏USP15-p53- r175h相互作用特异性地导致p53- r175h降解。这导致p53-R175H泛素化和突变蛋白的降解增加。基于这些发现，我们选择探索去泛素化酶（DUB）家族的其他成员作为突变型p53蛋白稳定性的潜在调节剂。在不同p53状态的卵巢癌细胞系中使用靶向dub的sgrna进行crispr干扰和crispr激活筛选，我们正在鉴定有助于突变型p53稳定性的dub。这些细胞系包括几种p53表达模型，p53-null、野生型和突变型p53- r175h，常见于卵巢癌。识别出的dub将作为可药物靶点进一步分析。选择性靶向dub的能力将是至关重要的，因为它们在泛素途径中起着多种作用，特别是通过关注与催化结构域相邻的n端结构域，这在不同的USPs中差异很大。目标是开发小分子和/或拟肽化合物，破坏突变p53的积累和/或恢复p53野生型肿瘤抑制功能。