Validating p53 Ser46 crotonylation as a potential target for possible anti-cancer therapy

验证 p53 Ser46 巴豆酰化作为可能的抗癌治疗的潜在靶点

基本信息

批准号：
10492834
负责人：
Hua Lu
金额：
$ 17.77万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-26 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10492834
关键词：
Acetic Acids Acetylation Acids Address Affect Alanine Bacteria Biological Biological Assay Biological Models Butyric Acids Cancer Cell Growth Cancer Etiology Cancerous Catalysis Cell Proliferation Cell model Cells Chemicals Chromatography Clustered Regularly Interspaced Short Palindromic Repeats Colon Carcinoma Colorectal Cancer Colorectal Neoplasms Coupled DNA Damage Defect Development Dietary Fiber EP300 gene Embryo Enzymes Equilibrium Essential Fatty Acids Fermentation Fibroblasts Functional disorder Future Gene Mutation Genes Genome Stability Growth Health Helicobacter pylori Histones Hot Spot Human Hypoxia Immunity Impairment In Vitro Individual Label Link Lysine Malignant Neoplasms Malignant neoplasm of lung Mediating Metabolic Microbe Modification Mus Mutate Mutation Nutrient Nutrient Depletion Oncogenic PCAF gene Pathway interactions Peptide antibodies Phosphorylation Physiology Play Post-Translational Protein Processing Propionic Acids Proteins Proteomics Reaction Reactive Oxygen Species Regulation Research Resistance Ribosomes Role Serine Solid Stress System TP53 gene Testing Transferase Tumor Suppressor Genes Ubiquitination Volatile Fatty Acids cancer cell cancer therapy colorectal cancer treatment epigenetic regulation fast protein liquid chromatography gut microbiome gut microbiota human disease insight microbiome microbiota mutant novel novel strategies novel therapeutics prevent response stressor tris(2-carboxyethyl)phosphine tumor

项目摘要

Project Summary Gut microbiota plays critical roles in maintaining human health, while their off-balance is highly associated with human diseases, including cancer. Some of them are linked to the tumor suppressor p53 pathway, such H. Pylori. p53 is important for maintaining genomic stability and preventing tumor formation in response to various stressors. Thus, its protein level and activity are tightly regulated via multiple mechanisms. Cancers also evolve different strategies to control p53 activity in favoring their growth and survival in addition to mutating its gene. A recent study showed that gut microbiome can convey the oncogenic function of a hot spot mutant p53. However, it remains unknown if and how gut microbiota might cause cancer by inactivating wild type p53 at the early stage of colorectal cancers (CRC), when p53 is rarely mutated. Recently, a type of unsaturated short chain fatty acid (SCFA) called crotonic acid (CA) has been shown to modify histone proteins for epigenetic regulations. However, CA has not been explored for p53 regulation till our recent study. CA is one of the common products of the dietary fiber fermentation by microflora in the human gut. Interestingly, when testing if CA could affect p53 level and activity by treating human wild type p53-containing CRC cells, we found that it can induce p53 crotonylation, but surprisingly reduce its protein, but not mRNA, levels in these cells. More surprisingly, this crotonylation targeted serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This was further confirmed by substitution of serine 46 with alanine (p53-S46A), which abolishes p53 crotonylation in vitro and in cells. CA increased p53- dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil a noncanonical PTM unique for human p53, impairing its activity in response to CA. Because CA is produced by the gut microbiome, we hypothesize that CA might play a critical role in early human colorectal neoplasia development by negating p53 activity without mutation of this gene. To test this hypothesis, we will first determine if Ser46 crotonylation plays a role in negating p53 activity in CRC cells and also purify the newly identified Ser46 crotonylation transferase from human cells by establishing two cell systems with p53 mutations at Ser46 or Pro47 and employing of FPLC chromatography coupled with proteomic analysis. Our studies will identify a novel enzyme that catalyzes p53 Ser46 crotonylation and provide new insights into how this noncanonical PTM regulates p53 level and activity in CRC cells. Importantly, completing these studies will open a new direction for studying the biological role of Ser- crotonylation in cancer development and offer proof-of-concept evidence for targeting this type of gut microbiota responsive posttranslational modifications, such as the newly identified crotonyltransferase or p53 Ser46 crotonylation, as a strategy for developing a new therapy for CRCs and possibly other solid cancers at their early stages when p53 is not mutated, but inactivated via crotonylation mediated by microbiota-produced CA.

项目摘要肠道微生物群在维持人类健康方面起着关键作用，而其不平衡与包括癌症在内的人类疾病。其中一些与肿瘤抑制p53途径有关，例如H。幽门螺杆菌。 p53对于维持基因组稳定性和防止各种肿瘤形成很重要压力源。因此，其蛋白质水平和活性通过多种机制严格调节。癌症也在发展除了突变其基因之外，还采用了控制p53活性以有利于其生长和生存的不同策略。一个最近的研究表明，肠道微生物组可以传达热点突变体p53的致癌功能。然而，肠道菌群是否以及如何通过在早期灭活野生型p53来引起癌症仍然未知 p53很少突变时结直肠癌（CRC）。最近，一种不饱和的短链脂肪酸（SCFA）称为子酸（CA）已证明可以修饰组蛋白进行表观遗传调节。然而，直到我们最近的研究，CA才被探讨了P53调节。 CA是人类肠道中的饮食纤维发酵。有趣的是，当测试CA是否会影响p53水平时和通过治疗含p53的CRC细胞的人类野生型CRC细胞的活性，我们发现它可以诱导p53 crotonylation，但令人惊讶的是，这些细胞中的蛋白质（而不是mRNA）降低了蛋白质。更令人惊讶的是，这种杂种靶向丝氨酸46，而不是p53的任何预测的赖氨酸残基，如TCEP探针所检测到的互链率和抗杂型肽抗体反应测定法。替代进一步证实了这一点与丙氨酸（p53-S46a）的丝氨酸46的含量，该甲氨酸在体外和细胞中废除了p53 crotonylation。 CA增加了P53- 依赖性糖酵解活性，并响应代谢或DNA损伤增强癌细胞增殖压力。由于丝氨酸46仅在人类p53中发现，因此我们的研究推出了人类独特的非规范性PTM p53，响应于ca而损害其活性。因为Ca是由肠道微生物组产生的，所以我们假设该CA可能通过否定p53活动而没有该基因的突变。为了检验这一假设，我们将首先确定Ser46 crotonylation是否在否定CRC细胞中的p53活性，并从中净化新鉴定的Ser46 crotonylation转移酶通过在Ser46或Pro47上建立两个具有p53突变的细胞系统并使用FPLC来建立人类细胞色谱与蛋白质组学分析结合。我们的研究将确定一种催化p53的新型酶 SER46共同化，并提供有关该非规范PTM如何调节p53水平和活动的新见解 CRC细胞。重要的是，完成这些研究将为研究服务的生物学作用打开新的方向癌症发育中的共同化并提供了针对此类肠道菌群的概念验证证据反应迅速的翻译后修饰，例如新确定的crotonyltransferase或p53 Ser46 杂种，作为一种为CRC和其他固体癌症开发新疗法的策略当p53未突变时，阶段是通过微生物群产生的ca介导的共同体灭活的。