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对应激反应的关键组成部分。它是由DNA损伤，缺氧，热休克和其他类型的压力激活的均序列特异性转录因子，可调节DNA修复，细胞周期停滞，衰老，代谢和凋亡。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的串联N末端反式激活结构域（TAD）对于p53作为转录因子至关重要。两个子域TAD1（残基1-40）和TAD2（残基35-59）与转录共激活因子p300的多个域相互作用。但是，这两个子域可以彼此独立地发挥作用，这表明参与了TAD1和TAD2的转录辅助因子在转录激活中的参与，其中相互作用可能受到p53磷酸化的差异调节。为了鉴定TAD1和TAD2的独特相互作用伴侣，分别合成且在N-Termerini的生物素合成，分别合成了THR 18或SER 46的肽（残基9-33）或TAD2（残基9-33）或TAD2（残基35-59）。我们将这些肽用作从用依托泊苷处理的MCF7细胞制备的核提取物的相互作用蛋白质的诱饵。肽的还原性二甲基化，然后进行质谱分析，以识别和定量比较相互作用者，以区分那些优先与TAD1或TAD2子域相互作用的人。我们使用生物学一式一式三份下拉的初步实验已经确定了潜在相互作用的列表，这些列表显示出对未经处理的细胞中未修饰或修饰的p53或依托泊苷处理后的偏爱。除了p53 TAD1和TAD2的已知结合伴侣外，我们还确定了几个新的交互剂。我们继续研究选定相互作用者对p53功能的影响。 p53的C末端表现出各种各样的PTM，包括磷酸化，甲基化，乙酰化，泛素化，Sumoylation，Neddylation和Neddylation和Hydroxylation，主要定位于蛋白质的末端三十个残基。我们已经表明，p53可以在LYS382上单甲基化和二甲基化，而前者的修饰抑制了p53的转录活性，后者促进了DNA修复，此外还证明了同一部位的乙酰化和泛素化。赖氨酸382上的SETD8单甲基酸盐p53，衰减p53促凋亡和生长停滞功能。与DRS合作，使用高含量成像siRNA屏幕和化学屏幕。 Veschi和Thiele，我们将SETD8确定为神经母细胞瘤细胞系中p53活性的抑制剂。 SETD8活性的遗传学或药理抑制作用通过降低p53K382Me1而导致p53野生型途径激活。最近，我们证明了在结肠癌干细胞中抑制setD8过表达导致p53的激活。我们正在开发高通量测定法，以鉴定IC50较低和体内耐受性较高的SETD8抑制剂。据报道，p53点突变发生在大约一半的人类肿瘤中，并且明显的特定“热点”残基的代表过多。这些突变废除了p53作为转录因子和肿瘤抑制因子的能力。此外，许多突变形式的p53已通过功能获取机制获得了新的致癌活动。许多热点p53突变体的一个特征是它们的结构不稳定性，部分展开和与淀粉样蛋白疾病中发现的聚集体的形成相似，从而导致蛋白质失活。最近，Padmanabhan等人。 （NatCommun。9（1）：1270（2018））报告说，去偶素酶抑制剂PR-619专门针对热点突变体P53-R175H以降级。 PR-619破坏了p53-R175H与去泛素酶，泛素特异性蛋白酶15（USP15）之间的相互作用，这导致了突变蛋白的泛素化和降解。根据这些发现，我们选择探索去泛素化酶（DUB）家族的其他成员作为突变体p53蛋白稳定性的潜在调节剂。我们的方法是使用DUB靶向SGRNA在卵巢癌细胞系中进行CRISPR干扰和CRIS-Activation筛查。我们希望确定有助于突变p53稳定性的配音，可以进一步审查为可毒靶。选择性靶向配音的能力将是至关重要的，因为它们在泛素途径中扮演多个角色。由于泛素特异性蛋白酶（USP）的催化结构域是高度保守的，因此必须探索催化结构域以外的区域以进​​行潜在的药物靶向。在USP之间，与催化结构域相邻的N末端结构域有显着变化，使这些有希望的区域可以找到独特的小分子结合口袋。最终，序列和结构数据可用于帮助开发小分子和/或肽型化合物的合理筛选，以找到更合适的候选物，以破坏突变体p53和/或救出p53野生型肿瘤抑制功能的积累。我们已经成功地表达了4个卵巢癌细胞系中的激活和抑制作用CRISPR-CAS9慢病毒质粒。这些线包括几种p53表达模型，p53-null，野生型和突变体p53-r175h，通常在卵巢癌中看到。此外，我们已经准备了靶标SGRNA的慢病毒库，并启动了我们的筛选实验。我们的未来目标是能够识别这些突变在各自肿瘤中可能存在的功能，代谢和转录差异，以及小分子的发展，肽症和基因治疗方法，以克服这些改变。