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. 2004 Nov;24(22):9958-67.
doi: 10.1128/MCB.24.22.9958-9967.2004.

NAD+ modulates p53 DNA binding specificity and function

Kevin G McLure  1 , Masatoshi Takagi, Michael B Kastan
Affiliations

NAD+ modulates p53 DNA binding specificity and function (VSports在线直播)

"V体育平台登录" Kevin G McLure et al. Mol Cell Biol. 2004 Nov.

Abstract

DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al. , Cell 71:587-597, 1992; K. H. Vousden and X VSports手机版. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD(+) binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B(3)) increases the rate of intracellular NAD(+) synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD(+) on p53, as a molecule structurally related to part of NAD(+), TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B(1)), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. .

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Figures

FIG. 1.
FIG. 1.
Structures of NAD+, ADP, and TDP.
FIG. 2.
FIG. 2.
Effect of NAD+ on sequence-specific DNA binding activity of recombinant p53 expressed in bacteria. (A) Wild-type human p53 was incubated with 32P-DNA sequences and electrophoresed under nondenaturing conditions. CON, p53 consensus binding site; NB, p53-nonbinding sequence containing mutations at four critical G/C contact residues; p21 and hdm2, 5′ p21 and hdm2 human genomic binding sites, respectively. Amino- and carboxy-terminal-specific DO1 and PAb421 monoclonal antibodies were added to supershift the p53-DNA complex. (B) p53 was incubated with 32P-hdm2 plus various nucleotides. In lane +Ab antibody PAb421 was added to supershift the p53-DNA complex. (C) NADH (5 mM) or NAD+ at the indicated concentrations were added as indicated. Sequence-specific binding controls include p53 plus the nonbinding control DNA sequence (lane NB) and hdm2 DNA without p53 (lane −p53). (D) Nucleotides were added as indicated to p53 and 32P-hdm2 DNA.
FIG. 3.
FIG. 3.
NAD+ binding, affecting p53 conformation and specifically inhibiting p53 tetramer DNA binding in vitro. (A, left) Equal amounts of p53 and L344A were incubated with 32P-NAD+. (Right) Bound 32P-NAD+. Signal strength is indicated below each spot; signal strength for anti-p53 is arbitrarily 100%. (B) [35S]methionine-labeled proteins were incubated with NAD+ or NADH and then digested with trypsin (tryp). The percentage of undigested p53 (arrow) is indicated below each lane. (C) NAD+ was added with 32P-hdm2 (top) or 32P-p21 (bottom) to p53 tetramers or dimers. DNA-bound proteins are tetramers (4×), single L344A dimers (2×), or pairs of L344A dimers (2-2×).
FIG. 4.
FIG. 4.
Effects of related small molecules on p53 DNA binding. (A) Portions of the NAD+ molecule were incubated with p53 and 32P-hdm2. NAm, niacinamide; FAD, flavin adenine dinucleotide. (B) Nucleoside diphosphates were added as indicated to p53-32P-hdm2 DNA. (C) Thiamine, TMP, or TDP was added to p53 DNA binding reactions. (D) Kinetic analysis of the dissociation of p53 from DNA in the presence of nucleotides as indicated. Nucleotides were added to p53-32P-hdm2, and then excess unlabeled hdm2 was added at time zero. The amount of p53 remaining bound to 32P-hdm2 at the indicated times is plotted. (E) The effect of NAD or TDP on p53 binding to p21 versus hdm2 DNA is quantified below each lane. NB and −p53 are as defined for Fig. 2.
FIG. 5.
FIG. 5.
Effects of niacinamide on p53 activity. (A) Quantification of intracellular NAD+ levels after treating MCF7 cells with niacinamide (NAm), irradiated (IR) as indicated to induce endogenous, wild-type p53. (B) Cells were treated with niacinamide to increase NAD+ and then irradiated to induce endogenous, wild-type p53. After 3 h (lane IR), genomic DNA sites that were bound to p53 protein were analyzed by ChIP (IP). Ctrl, control. (C) Cells were treated as in panel B, but RNA was extracted and analyzed by semiquantitative reverse transcription-PCR. (D) Cells were untreated or treated with niacinamide and then irradiated and allowed to recover for 1, 3 or 5 h (IR1, IR3, and IR5, respectively). Proteins were then extracted and analyzed by Western blotting. (E) MCF7 cells were incubated with 2 or 5 mM niacinamide and then Western blotted for total p53 or the loading control brg in whole-cell extract (lower two panels) or for immunoprecipitated total or K373/K382 acetylated (Ac) p53 (upper two panels). (F) Western blot of p53 or hdm2 in whole-cell extracts prepared before the onset of p53 degradation due to hdm2 and at 1.5 and 2.5 h after 6-Gy irradiation.
FIG. 6.
FIG. 6.
Effects of thiamine on p53 activity (A) Cells were treated with thiamine (Thia) to increase TDP and then irradiated (IR) to induce endogenous, wild-type p53. After 3 h (lane IR), genomic DNA sites that were bound to p53 protein were analyzed by ChIP (IP). (B) Cells were untreated or treated with thiamine and then irradiated and allowed to recover for the 3 or 5 h (IR3 and IR5, respectively). Proteins were then extracted and analyzed by Western blotting. (C) In the same experiment, cells were treated with niacinamide (NAm) or thiamine, and then proteins were extracted and analyzed by Western blotting. (D) Immunoprecipitated p53 was Western blotted for total p53 (bottom) or K373/K382 acetylated (Ac) p53 (top) after treatment with thiamine or niacinamide, followed by irradiation.
FIG. 7.
FIG. 7.
Effects of increased NAD+ or TDP on cellular responses to ionizing radiation. (A) MCF7 cells were treated to increase NAD+ or TDP and then irradiated (IR). After 5 days, chromosome aneuploidy in 100 metaphase spreads per sample was analyzed. Bars indicate the numbers of cells displaying aneuploidy greater than that displayed by the average control cell. NAm, niacinamide; Cont, control. (B) Murine thymocytes from wild-type (p53+/+) or p53 knockout (p53−/−) mice were explanted and incubated to increase NAD+ or TDP and then irradiated. Dex, dexamethasone treatment. (C) Mice were treated to increase NAD+ and then irradiated. Thymocytes were collected after 18 h.
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References

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