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

项目摘要 肠道微生物区系在维持人类健康方面起着至关重要的作用，而它们的失衡与 人类疾病，包括癌症。其中一些基因与肿瘤抑制因子P53途径有关，如H。 幽门螺杆菌。P53在维持基因组稳定性和防止肿瘤形成方面具有重要意义 压力源。因此，它的蛋白质水平和活性受到多种机制的严格调控。癌症也会进化 控制P53活性的不同策略除了突变其基因外，还有利于它们的生长和存活。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区系是否以及如何通过在早期阶段使野生型p53失活而导致癌症。 在结直肠癌(CRC)中，p53很少发生突变。最近，一种不饱和短链脂肪酸 被称为巴豆酸(CA)的单链脂肪酸(SCFA)已被证明可以修饰组蛋白以实现表观遗传调节。然而， 直到我们最近的研究才发现CA对P53的调控作用。CA是通用的产品之一， 人体肠道微生物区系发酵膳食纤维。有趣的是，当测试CA是否会影响P53水平时 和活性通过处理人野生型P53-CRC细胞，我们发现它可以诱导P53巴豆化， 但令人惊讶的是，它降低了这些细胞中的蛋白质水平，而不是信使核糖核酸水平。更令人惊讶的是，这种巴豆化 靶向丝氨酸46，而不是任何预测的赖氨酸残基，如在标记的TCEP-探针中检测到的 巴豆化和抗巴豆化多肽抗体反应检测。这一点通过替换得到了进一步的证实 丝氨酸46与丙氨酸(P53-S46A)的结合，在体外和细胞内取消了P53的巴豆化。CA增加P53- 依赖糖酵解活性，并在代谢或DNA损伤时促进癌细胞增殖 压力。由于丝氨酸46只在人类p53中发现，我们的研究揭示了一种人类独有的非规范的PTM P53对CA的应答作用减弱其活性。因为CA是由肠道微生物群产生的，所以我们假设 CA可能通过否定P53活性而在早期人类结直肠肿瘤的发生发展中发挥关键作用 这种基因的突变。为了验证这一假设，我们将首先确定Ser46巴豆化是否在 在结直肠癌细胞中否定P53活性并纯化新发现的Ser46巴豆化转移酶 P53基因Ser46和Pro47突变人细胞系的建立及FPLC的应用 层析与蛋白质组分析相结合。我们的研究将确定一种新的催化P53的酶 Ser46巴豆化，为这种非规范的PTM如何调节P53水平和活性提供了新的见解。 CRC信元。重要的是，完成这些研究将为研究丝氨酸的生物学作用开辟新的方向。 巴豆化在癌症发展中的作用并为以这种肠道微生物区系为靶点提供概念验证证据 有反应性的翻译后修饰，如新发现的巴豆酰转移酶或P53 Ser46 巴豆化作为一种策略，用于开发一种新的疗法来治疗早期的癌和可能的其他实体癌 P53没有突变，而是通过微生物产生的CA介导的巴豆化而失活的阶段。

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.