Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

• 批准号: 10262014
• 负责人: ETTORE APPELLA
• 金额: $ 57.98万
• 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/10262014
• 关键词: Acetylation; Affect; Amyloidosis; Apoptosis; Apoptotic; Attenuated; Binding; Biochemical; Biological; Biotin; C-terminal; CRISPR interference; CRISPR/Cas technology; Cancer cell line; Catalytic Domain; Cell Cycle Arrest; Cell Line; Cells; Characteristics; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colon Carcinoma; DNA Damage; DNA Repair; Deubiquitinating Enzyme; Development; Drug Targeting; EP300 gene; Etoposide; Exhibits; Family; Future; Genetic; Genetic Transcription; Goals; Growth; Heat-Shock Response; Homo; Hot Spot; Human; Hydroxylation; Hypoxia; Image; Libraries; Lysine; MCF7 cell; Malignant neoplasm of ovary; Mass Spectrum Analysis; Metabolic; Metabolism; Methods; Methylation; Modeling; Modification; Molecular Conformation; Mutation; N-terminal; Nuclear Extract; Nuclear Proteins; Oncogenic; Outcome; Pathway interactions; Peptides; Pharmacology; Phosphorylation; Plasmids; Point Mutation; Post-Translational Protein Processing; Protein p53; Proteins; Regulation; Reporting; Research; Role; Site; Small Interfering RNA; Stress; Structure; Subfamily lentivirinae; TP53 gene; Transactivation; Transcription Coactivator; Transcriptional Activation; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; anticancer treatment; base; cancer stem cell; cofactor; differential expression; druggable target; experimental study; gain of function; gene therapy; high throughput screening; improved; in vivo; inhibitor/antagonist; member; mutant; neuroblastoma cell; novel; overexpression; peptidomimetics; preference; response; screening; senescence; small molecule; structured data; transcription factor; tumor; ubiquitin-specific protease

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's 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; reductive dimethylation of peptides followed by 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 preliminary 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 continue to investigate the effect of selected interactors on the function of p53. 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. Recently, we demonstrated that inhibition of SETD8 overexpression in colon cancer stem cells results in the activation of p53. We are developing high-throughput assays to identify inhibitors of SETD8 with a lower IC50 and higher 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. One characteristic of many of the hot-spot p53 mutants is their structural instability with partial unfolding and the formation of aggregates similar to those seen in amyloid diseases, thus resulting in protein inactivation. Recently, Padmanabhan et al. (Nat Commun. 9(1):1270 (2018)) reported that the deubiquitinase inhibitor, PR-619, specifically targets the hot-spot mutant p53-R175H for degradation. PR-619 disrupts the interaction between p53-R175H and a deubiquitinase, ubiquitin-specific protease 15 (USP15), which leads to 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. Our approach is to conduct CRISPR-interference and CRISPR-activation screens in ovarian cancer cell lines with varying p53 status using DUB-targeting sgRNAs. We hope to identify DUBs that contribute to mutant p53 stability that can be further scrutinized as druggable targets. The ability to selectively target DUBs will be crucial since they take on several roles in the ubiquitin pathway. As the catalytic domains of Ubiquitin-specific proteases (USPs) are highly conserved, regions outside the catalytic domain must be explored for potential drug targeting. The N-terminal domains adjacent to the catalytic domain vary significantly among USPs, making these promising regions for finding unique small molecule-binding pockets. Eventually, sequence and structure data can be used to help develop a rational screen of small molecule and/or peptidomimetic compounds to find more suitable candidates to disrupt the accumulation of mutant p53 and/or rescue p53 wildtype tumor suppressive function. We have successfully expressed both activation and inhibition CRISPR-Cas9 lentivirus plasmids in 4 ovarian cancer cell lines. These lines encompass several p53 expression models, p53-null, wild-type and mutant p53-R175H, commonly seen in ovarian cancer. Additionally, we have prepared lentivirus libraries of DUB-targeting sgRNAs and have initiated our screening experiments. Our future aims are to be able to recognize the functional, metabolic and transcriptional differences these mutations can have in their respective tumors, and the development of small molecules, peptidomimetics and gene therapy methods to overcome these alterations.

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。p53的串联n端反激活结构域（TADs）对p53作为转录因子的活性至关重要。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；利用肽的还原二甲基化和质谱分析来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初步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处理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。我们将继续研究选定的相互作用物对p53功能的影响。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。最近，我们证明了在结肠癌干细胞中抑制SETD8过表达会导致p53的激活。我们正在开发高通量检测方法，以鉴定具有较低IC50和较高体内耐受性的SETD8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。许多热点p53突变体的一个特征是它们的结构不稳定，部分展开和形成聚集体类似于淀粉样蛋白疾病，从而导致蛋白质失活。最近，Padmanabhan等人(Nat commin . 9（1):1270(2018)）报道了去泛素酶抑制剂PR-619特异性靶向热点突变体p53-R175H进行降解。PR-619破坏p53-R175H与去泛素酶、泛素特异性蛋白酶15 （USP15）之间的相互作用，导致突变蛋白泛素化和降解。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。我们的方法是使用靶向dub的sgRNAs在具有不同p53状态的卵巢癌细胞系中进行crispr干扰和crispr激活筛选。我们希望确定dub有助于突变p53的稳定性，可以进一步仔细研究作为药物靶点。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扮演着几个角色。由于泛素特异性蛋白酶（USPs）的催化结构域是高度保守的，因此必须探索催化结构域外的区域作为潜在的药物靶向。与催化结构域相邻的n端结构域在不同的USPs中差异很大，使得这些有希望找到独特的小分子结合口袋的区域。最终，序列和结构数据可用于帮助开发小分子和/或拟肽化合物的合理筛选，以找到更合适的候选物来破坏突变型p53的积累和/或恢复p53野生型肿瘤抑制功能。我们在4种卵巢癌细胞系中成功表达了激活和抑制CRISPR-Cas9慢病毒质粒。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。此外，我们已经准备了针对dub的sgrna慢病毒文库，并开始了筛选实验。我们未来的目标是能够识别这些突变在各自肿瘤中的功能、代谢和转录差异，并开发小分子、拟肽和基因治疗方法来克服这些改变。