Functional analysis of the p300 acetyltransferase domain: the PHD finger of p300 but not of CBP is dispensable for enzymatic activity

Acetylation of nucleosomal histones is a major regulatory step during activation of eukaryotic gene expression. Among the known acetyltransferase (AT) families, the structure–function relationship of the GNAT superfamily is the most well understood. In contrast, less information is available regarding mechanistic and regulatory aspects of p300/CBP AT function. In this paper, we investigate in closer detail the structure and sequence requirements for p300/CBP enzymatic activity. Unexpectedly, we find that the PHD finger of p300, but not of CBP, is dispensable for AT activity. In order to identify residues involved in substrate or acetyl-coenzyme A (acetyl-CoA) recognition, we have introduced 19 different amino acid substitutions in segments that are highly conserved between animal and plant p300/CBP proteins. By performing acetylation reactions with histones, a p53 peptide or the AT domain itself, we define several residues required for histone and p53 substrate recruitment but not for acetyl-CoA binding. Finally, we show that identical mutations in the p300 and CBP AT domain impair AT activity differently. This latter result combined with the finding of a differential requirement for the PHD finger provides evidence for structural differences between p300 and CBP that may in part underlie a previously reported functional specialization of the two proteins.     

The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.     

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.