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Issue 1, 2011

Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen® technology

Author affiliations

Abstract

Upon genomic insult, the tumor suppressor p53 is phosphorylated and acetylated at specific serine and lysine residues, increasing its stability and transactivation function. Deacetylases, including the type III histone deacetylase SIRT1, remove acetyl groups from p53 and counterbalance acetyltransferase activity during a DNA damage response. This report describes a series of high-throughput LanthaScreen® time-resolved Förster resonance energy transfer (TR-FRET) immunoassays for detection of intracellular p53 phosphorylation of Ser15 and acetylation of Lys382 upon treatment with DNA damage agents, such as etoposide. These assays were used to measure the deacetylase activity of SIRT1 and/or Type I/II Histone deacetylases (HDACs). First, BacMam-mediated overexpression of SIRT1 resulted in dose-dependent deacetylation of GFP-p53 following etoposide treatment of U-2 OS cells, confirming that GFP-p53 serves as a SIRT1 substrate in this assay format. Further, overexpression of the acetyltransferase p300 via BacMam increased the acetylation of GFP-p53 at Lys382. Next, siRNA-mediated knockdown of SIRT1 resulted in increased GFP-p53 acetylation, indicating that endogenous SIRT1 activity can also be measured in U-2 OS cells. Consistent with these results, GFP-p53 acetylation was also increased upon treatment of cells with a small-molecule inhibitor of SIRT1, EX-527. The effect of this compound was dramatically increased when used in combination with chemotherapeutic drug and/or the HDAC inhibitor Trichostatin A, confirming a proposed synergistic mechanism of p53 deacetylation by SIRT1 and Type I/II HDACs. Taken together, the cellular assays described here can be used as high-throughput alternatives to traditional immunoassays such as western blotting for identifying pharmacological modulators of specific p53-modifying enzymes.

Graphical abstract: Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen® technology

Supplementary files

Article information


Submitted
28 May 2010
Accepted
13 Sep 2010
First published
08 Oct 2010

Mol. BioSyst., 2011,7, 59-66
Article type
Paper

Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen® technology

M. B. Robers, C. Loh, C. B. Carlson, H. Yang, E. A. Frey, S. B. Hermanson and K. Bi, Mol. BioSyst., 2011, 7, 59 DOI: 10.1039/C0MB00026D

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