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 typ...