%0 Journal Article
%T p53 ¦Â-hydroxybutyrylation attenuates p53 activity
%A Fangzhou Li
%A Feng Tian
%A Haichao Han
%A Kaiqiang You
%A Kun Liu
%A Meiyu Geng
%A Ning Lin
%A Qianqian Sun
%A Tanjun Tong
%A Tingting Li
%A Wei Gu
%A Wenhui Zhao
%A Yingming Zhao
%A Zebin Mao
%J Archive of "Cell Death & Disease".
%D 2019
%R 10.1038/s41419-019-1463-y
%X a kbhb modification of overexpressed p53 in 293T cells. Flag-p53 was purified from 293T cells treated with 10£¿mM ¦Â-hydroxybutyrate (BHB), pyruvate or glutamine for 24£¿h. The purified protein was detected by western blot using anti-pan ¦Â-hydroxybutyrylation-lysine antibodies (BHB-K) or anti-p53 (DO-1). b Kbhb modification of endogenous p53 in U2OS cells. Western blot with p53 (DO-1), BHB-K or actin antibodies of cell lysates of U2OS cells treated with 10£¿mM BHB (bottom half, labeled as ¡°lysate¡±) or cell lysates immunoprecipitated (IP) with BHB-K antibodies (top, IP: BHB-K) with ReliaBLOT£¿ IP/Western Blot Reagent (Bethyl) to inhibit the signal of the Ig heavy chain interference. c Kbhb modification of endogenous p53 in HCT116 cells. Cell lysates of HCT116 cells treated with or without 10£¿mM BHB for 24£¿h were IP with BHB-K antibodies. Western blot analysis of p53 by DO-1 antibodies as in (b). d The BHB in mouse serum were measured. Mice were fasted for 48£¿h or fed with a normal diet. Blood samples were taken from a tail vein for determination of blood BHB concentrations using a ketone meter. e Kbhb modification of endogenous p53 in mice thymus tissue. Thymus cell lysates of mice fasted for 48£¿h or fed with a normal diet were IP with BHB-K antibodies. Western blot analysis of p53 with anti-full-length p53 antibodies as in (b). f£¿h Mass spectrometry analysis identified p53-derived peptides containing ¦Â-hydroxybutyrylated Lys-120, Lys-319, and Lys-370. p53 purified from 293T cells transfected with plasmid DNA expressing Flag-p53 and treated with 10£¿mM BHB for 24£¿h was analyzed by HPLC-MS/MS. Annotated MS/MS spectra of peptides LGFLHSGTAKSVTCTYSPALMK (derived from amino acids 111£¿132, f), ALNTSSSPQPKK (derived from amino acids 307£¿320, g) and AHSSSHLKSK (derived from amino acids 364£¿372, h) was showing as b- and y-ion coverage. i Effect of lysine-to-arginine mutation on p53 kbhb. Western blot analysis using BHB-K of wild-type and mutant Flag-p53 expressed in 293T cells treated with 10£¿mM BHB for 24£¿
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411878/