Crotonylation at serine 46 impairs p53 activity
Introduction
p53 is pivotally important for maintaining genomic stability and preventing tumor formation in response to various stressors, such as DNA damage, reactive oxygen species (ROS), ribosomal stress, nutrient depletion, hypoxia and oncogenic overloading [1]. Yet, when overly or abnormally activated, p53 can cause developmental defects and human diseases [2]. 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 [3], such as modifying its protein [4] in addition to mutating its gene, as it is the most frequently inactivated protein in all types of human cancers [5]. In fact, p53 can be modified via various forms of PTM, which either positively (acetylation or phosphorylation) or negatively (ubiquitination) regulate p53 stability and activity [6]. Recently, a type of unsaturated short chain fatty acid (SCFA) called Crotonic acid (CA), has been shown to modify histone proteins for epigenetic regulations [7]. However, this type of modifications has not been explored for p53 regulation.
Section snippets
Plasmids and antibodies
The His-tagged p53 was generated into the pET30a vector, and His-p53-S46A was mutant based on His-p53. The no-tag plasmid pcDNA3.1-p53, pcDNA3.1-p53-S33A, pcDNA3.1-p53-S46A, pcDNA3.1-p53-T81A, pcDNA3.1-p53-4 M were obtained from Giannino Del Sal. p53-K24R was mutant based on pcDNA3.1-p53. The Flag-pcDNA-p53, Flag-pcDNA-p53-1-300, Flag-pcDNA-p53-101-300, Flag-pcDNA-p53-101-393 were obtained from Mu-shui Dai. pcDNA-p53-8KR was obtained from Wei Gu. The following antibodies were commercially
Crotonic acid negatively regulates p53 level and activity
SCFAs, including acetic acid (AA), propionic acid (PA), butyric acid (BA), and CA whose synonym is but-2-enoic acid (Fig. 1A), are common products of the dietary fiber fermentation by microflora in the human gut. Since these SCFAs are essential for molecule functions and human health, we first tested whether they would affect p53 level and activity by treating human wild type (wt) p53-containing colon cancer RKO or lung cancer H460 cells with each of the acids individually. Interestingly, PA or
Discussion
As presented above, our study unveils crotonylation as a new type of PTM for p53 regulation in response to CA treatment. This finding is novel as it is the first time to learn that crotonylation can occur at a serine residue, instead of lysine, of a protein (Figs. 2 and S2). Also, by crotonylating serine 46, CA can negatively regulate p53 level and activity and consequently render the wt p53-dependent resistance of cancer cells to chemotherapeutic drugs or nutrient stress (Fig. 1, Fig. 3, Fig. 4
Author contributions
P.L. conducted the majority of the experiments, analyzed the results and organized them into figures under supervision of S.X.Z. and H.L.; N.B. assisted with in vitro crotonylation of p53 experiment; B.C. assisted with ubiquitin of p53 experiment; X.Z, and J.H.J., assisted with CA mediated p53 resistance on drug treatment experiment; S.X.Z assisted with isotope label crotonylation experiment; K.D., S.T., and B.W., synthesized crotonylated serine; T.F., and W.C.W. synthesized crotonylated
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
We thank Wei Gu, Mu-shui Dai and Giannino Del Sal for providing plasmids, Zhiqiang Qin and Heather Machado for offering some cancer cell lines, as well as all of the Lu lab members for active discussion. Hua Lu and Shelya X Zeng were supported in part by National Institutes of Health (NIH)-National Cancer Institute (NCI) grants (R01CA095441, R01CA172468, R01CA127724). Binghe Wang was partially supported by the NIH-NCI grant (R01-CA180519).
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