Crotonylation at serine 46 impairs p53 activity

https://doi.org/10.1016/j.bbrc.2020.01.152Get rights and content

Highlights

  • p53 in human cells is crotonylated in response to crotonic acid.

  • This crotonylation is detected independently by a Pan anti-crotonylated peptide antibody and a TCEP probe.

  • This crotonylation surprisingly occurs at the serine 46 residue of p53.

  • Substitution of serine 46, but not any of predicted lysines, by alanine abolishes this p53 crotonylation.

  • Crotonic acid increases glycolytic activity and cancer cell proliferation by impairing p53 activity in human cancer cells.

Abstract

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets 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 is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases 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 an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.

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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