Targeting of Histone Acetyltransferase p300 by Cyclopentenone Prostaglandin 螖12-PGJ2 through Covalent Binding to Cys1438

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• 作者：Kodihalli C. Ravindra ; Vivek Narayan ; Gerald H. Lushington ; Blake R. Peterson ; K. Sandeep Prabhu
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：February 20, 2012
• 年：2012
• 卷：25
• 期：2
• 页码：337-347
• 全文大小：515K
• 年卷期：v.25,no.2(February 20, 2012)
• ISSN：1520-5010

文摘

Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys¹⁴³⁸ in the substrate binding site could be targeted by 螖¹²-prostaglandin J₂ (螖¹²-PGJ₂), a cyclopentenone prostaglandin (CyPG) derived from PGD₂. We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the 伪,尾-unsaturation in the cyclopentenone ring of 螖¹²-PGJ₂ was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-螖^12,14-PGJ₂, which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that 螖¹²-PGJ₂ places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys¹⁴³⁸ of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of 螖¹²-PGJ₂ with Cys¹⁴³⁸. Using biotinylated derivatives of 螖¹²-PGJ₂ and 9,10-dihydro-15-deoxy-螖^12,14-PGJ₂, we demonstrate the covalent interaction of 螖¹²-PGJ₂ with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, 螖¹²-PGJ₂ also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that 螖¹²-PGJ₂ inhibits p300 through Michael addition, where 伪,尾-unsaturated carbonyl function is absolutely required for the inhibitory activity.