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.

项目概要肠道微生物群在维持人类健康方面发挥着关键作用，而它们的失衡与人类疾病，包括癌症。其中一些与肿瘤抑制因子 p53 通路有关，例如 H. 幽门螺杆菌。 p53 对于维持基因组稳定性和防止肿瘤形成以应对各种情况非常重要压力源。因此，其蛋白质水平和活性通过多种机制受到严格调节。癌症也会进化除了突变其基因之外，还采用不同的策略来控制 p53 活性，以促进其生长和生存。一个最近的研究表明，肠道微生物组可以传递热点突变体 p53 的致癌功能。然而，目前尚不清楚肠道微生物群是否以及如何通过在早期阶段灭活野生型 p53 来导致癌症当 p53 很少发生突变时，结直肠癌 (CRC) 的发生率就会升高。最近，一种不饱和短链脂肪酸被称为巴豆酸 (CA) 的 SCFA 已被证明可以修饰组蛋白以进行表观遗传调控。然而，直到我们最近的研究为止，CA 还没有被探索过对 p53 的调节。 CA是常见产品之一膳食纤维通过人体肠道微生物发酵。有趣的是，在测试 CA 是否会影响 p53 水平时通过处理含有野生型 p53 的 CRC 细胞，我们发现它可以诱导 p53 巴豆酰化，但令人惊讶的是，它降低了这些细胞中蛋白质的水平，但没有降低 mRNA 的水平。更令人惊讶的是，这种巴豆酰化目标丝氨酸 46，而不是 p53 的任何预测的赖氨酸残基，如在标记的 TCEP 探针中检测到的巴豆酰化和抗巴豆酰化肽抗体反应测定。通过替换进一步证实了这一点丝氨酸 46 与丙氨酸 (p53-S46A) 的结合，可在体外和细胞内消除 p53 巴豆酰化。 CA 增加 p53- 依赖糖酵解活性，并增强癌细胞增殖以应对代谢或 DNA 损伤压力。由于丝氨酸 46 仅在人类 p53 中发现，因此我们的研究揭示了人类独有的非规范 PTM p53，削弱其响应 CA 的活性。因为 CA 是由肠道微生物组产生的，我们假设 CA可能通过在不影响p53活性的情况下消除p53活性而在早期人类结直肠肿瘤的发展中发挥关键作用该基因的突变。为了检验这个假设，我们首先确定 Ser46 巴豆酰化是否在消除 CRC 细胞中的 p53 活性，并纯化新鉴定的 Ser46 巴豆酰化转移酶通过建立两个在 Ser46 或 Pro47 处具有 p53 突变的细胞系统并采用 FPLC 来检测人类细胞色谱法与蛋白质组分析相结合。我们的研究将鉴定一种催化 p53 的新型酶 Ser46 巴豆酰化，并提供关于这种非规范 PTM 如何调节 p53 水平和活性的新见解 CRC 细胞。重要的是，完成这些研究将为研究 Ser- 的生物学作用开辟新的方向。巴豆酰化在癌症发展中的作用，并为针对此类肠道微生物群提供概念验证证据响应性翻译后修饰，例如新鉴定的巴豆酰转移酶或 p53 Ser46 巴豆酰化，作为开发针对 CRC 和可能的其他早期实体癌症的新疗法的策略 p53 未突变，但通过微生物产生的 CA 介导的巴豆酰化而失活的阶段。