Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

Lu Yang,Yuesheng Zhang,Arup Bhattacharya,Yun Li

doi:10.1038/s42003-021-02880-x

Abstract

Tumor suppressor p53, a critical regulator of cell fate, is frequently mutated in cancer. Mutation of p53 abolishes its tumor-suppressing functions or endows oncogenic functions. We recently found that p53 binds via its proline-rich domain to peptidase D (PEPD) and is activated when the binding is disrupted. The proline-rich domain in p53 is rarely mutated. Here, we show that oncogenic p53 mutants closely resemble p53 in PEPD binding but are transformed into tumor suppressors, rather than activated as oncoproteins, when their binding to PEPD is disrupted by PEPD knockdown. Once freed from PEPD, p53 mutants undergo multiple posttranslational modifications, especially lysine 373 acetylation, which cause them to refold and regain tumor suppressor activities that are typically displayed by p53. The reactivated p53 mutants strongly inhibit cancer cell growth in vitro and in vivo. Our study identifies a cellular mechanism for reactivation of the tumor suppressor functions of oncogenic p53 mutants.

Highlights

Tumor suppressor p53, a critical regulator of cell fate, is frequently mutated in cancer
We recently found that peptidase D (PEPD), known as prolidase, binds to the proline-rich domain (PRD) in p53 and PEPD knockdown (KD) activates p53 by disrupting its binding to PEPD7
We measured PEPD binding to p53 mutants by enzyme-linked immunosorbent assay (ELISA). p53 was included for comparison. p53mPRD, in which 11 of the 12 prolines in the PRD are replaced by alanines, does not bind to PEPD7 and was used as a negative control to confirm the specificity of the ELISA

Summary

Results

PEPD siRNA activated multiple caspases (caspase 9, 8, 7) in all the cell lines except MDA-MB-231 (p53KO) cells (Fig. 3b; Supplementary Fig. 4a, b) These results suggest that PEPD KD reactivates p53 mutants. If MDA-MB-231 (p53KO) cells transfected with a p53 mutant were treated with PEPD siRNA, cell survival was markedly inhibited (Fig. 4b), which was accompanied by no change in p53 mutant expression, marked PEPD KD, induction of CD95, PUMA and p21, and activation of caspase 7, but no increase or slight decrease in MYC, EGFR, and MKK3 (Fig. 4c). Focusing on p53R175H in SK-BR-3 cells, we showed that MDM2 KD by siRNA blocks PEPD KD-induced mitochondrial enrichment of p53R175H (Supplementary Fig. 6a) and that PEPD KD increases MDM2 binding to p53R175H and enriches mono-ubiquitinated p53R175H in a Relative cell survival. The in vivo tumorsuppressing activities of p53R175H, p53R248Q, and p53R280K reactivated by PEPD KD are similar to that of p53 activated by PEPD KD

Discussion

Findings

Methods