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

项目摘要 肠道微生物群在维持人类健康方面起着至关重要的作用，而它们的失衡与以下因素高度相关： 人类疾病，包括癌症。其中一些与肿瘤抑制基因p53通路有关，如H. Pylori p53对于维持基因组稳定性和预防肿瘤形成是重要的， 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 不同的策略来控制p53活性，除了使其基因突变之外，还有利于它们的生长和存活。一 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，在这方面， 目前尚不清楚肠道微生物群是否以及如何通过在早期灭活野生型p53而导致癌症。 结直肠癌（CRC），当p53很少突变。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节。然而，在这方面， 直到我们最近的研究，CA还没有探索p53调节。CA是通用的产品之一， 通过人体肠道中的微生物菌群发酵膳食纤维。有趣的是，当测试CA是否会影响p53水平时， 和活性，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的蛋白质水平，而不是mRNA水平。更令人惊讶的是， 靶向丝氨酸46，而不是任何预测的赖氨酸残基，p53，如在TCEP标记的探针中检测到的 巴豆酰化和抗巴豆酰化肽抗体反应测定。这一点通过替代得到了进一步证实 丝氨酸46与丙氨酸（p53-S46 A），其在体外和细胞中消除p53巴豆酰化。CA增加p53 依赖性糖酵解活性，并增加癌细胞增殖，以响应代谢或DNA损伤 应力由于丝氨酸46只存在于人p53中，我们的研究揭示了一个非经典的PTM， p53，削弱其响应CA的活性。由于CA是由肠道微生物组产生的，我们假设 CA可能通过抑制p53活性而在早期人类结直肠肿瘤发展中起关键作用， 这个基因的突变。为了验证这一假设，我们将首先确定Ser 46巴豆酰化是否在 消除CRC细胞中的p53活性，并且还从CRC细胞中纯化新鉴定的Ser 46巴豆酰化转移酶， 通过建立p53突变的Ser 46或Pro47两种细胞系统， 层析结合蛋白质组学分析。我们的研究将确定一种新的酶，催化p53 Ser 46巴豆酰化，并提供了新的见解，这种非经典的PTM如何调节p53水平和活性， CRC细胞。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。 巴豆酰化在癌症发展中的作用，并为靶向这种类型的肠道微生物群提供概念验证证据 响应性翻译后修饰，如新鉴定的巴豆酰转移酶或p53 Ser 46 巴豆酰化，作为开发CRCs和可能的其他早期实体癌的新疗法的策略， p53未突变，但通过微生物群产生的CA介导的巴豆酰化失活。

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.