The ability of p53 to induce apoptosis plays an important role in tumor suppression. Here, we describe a previously unknown posttranslational modification of the DNA-binding domain of p53. This modification, acetylation of lysine 120 (K120), occurs rapidly after DNA damage and is catalyzed by the MYST family acetyltransferases hMOF and TIP60. Mutation of K120 to arginine, as occurs in human cancer, debilitates K120 acetylation and diminishes p53-mediated apoptosis without affecting cell-cycle arrest. The K120R mutation selectively blocks the transcription of proapoptotic target genes such as BAX and PUMA while the nonapoptotic targets p21 and hMDM2 remain unaffected. Consistent with this, depletion of hMOF and/or TIP60 inhibits the ability of p53 to activate BAX and PUMA transcription. Furthermore, the acetyllysine 120 (acetyl-K120) form of p53 specifically accumulates at proapoptotic target genes. These data suggest that K120 acetylation may help distinguish the cell-cycle arrest and apoptotic functions of p53.

Summary The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. While p300 inhibitors have been reported, a potent, selective, and readily available active-site directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a Ki of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anti-cancer target.

Rtt109, also known as KAT11, is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genome stability. Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity. Here we report the X-ray crystal structure of an Rtt109–acetyl coenzyme A complex and carry out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109–Vps75 complex and studies of Rtt109 function in vivo. The Rtt109 structure reveals noteworthy homology to the metazoan p300/CBP HAT domain but exhibits functional divergence, including atypical catalytic properties and mode of cofactor regulation. The structure reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells. Implications for understanding histone substrate and chaperone binding by Rtt109 are discussed.

Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetylLys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific anti-acetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation. We also use this approach to obtain functional evidence that acetylation of CK2 protein kinase on Lys102 can stimulate its catalytic activity.Reversible acetylation of histones catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is recognized as a central mechanism of chromatin regulation. Beyond histones, Lys acetylation has been observed in more than 2500 mammalian proteins on more than 4000 sites and has been shown to govern many biological processes.1 Several approaches are available to explore the structural and functional consequences of protein acetylation. Traditional site-directed mutagenesis is used to replace Lys with Arg or Gln to provide indirect insights into Lys acetylation. Expressed protein ligation and unnatural amino acid mutagenesis via nonsense suppression can install acetylLys (AcK) at desired protein sites but have various technical limitations.2 pcole@jhmi.edu. Supporting Information Available. Detailed experimental procedures and Figures S1-S10. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 July 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAs an analog of methyl-Lys, methyl-thiaLys can be introduced at targeted protein locations via a relatively simple strategy involving Cys alkylation with N-methyl-aminoethylbromide derivatives.3 To create an acetyl-thiaLys analog (Figure 1a), we attempted to use the corresponding acetamide reagent with Cys-containing peptide; however, no significant conversion was observed. We hypothesized that the reduced ability of the amide compounds to form three-membered ring intermediates and/or intramolecular imidate formation led to diminished activity of N-acetyl-aminoethylbromide. We then explored the reaction of Nacetyl-aziridine and a Cys-containing peptide (Figure 1b). The predominant product observed by mass spectrometry was M+42 suggesting that acetyl-aziridine treatment led to an undesired acetyl transfer by nucleophilic attack at the carbonyl rather than the aziridine methylene ( Figure S1).To reduce reactivity at the carbonyl, we explored methylthiocarbonyl-aziridine (MTCA) (Figure 1b).4 Cys alkylation with this reagent was proposed to provide methylthiocarbonylthiaLys (MTCTK), a thiocarbama...